Targeted Degradation of the Myc Oncogene Using Pp2a

TARGETED DEGRADATION OF THE MYC ONCOGENE USING PP2A-

B56ALPHA SELECTIVE SMALL MOLECULE MODULATORS OF PROTEIN

PHOSPHATASE 2A AS A THERAPEUTIC STRATEGY FOR TREATING MYC-

DRIVEN CANCERS

CAROLINE C. FARRINGTON

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Thesis Advisor: Dr. Goutham Narla, M.D., Ph.D.

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

May 2020 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Caroline Farrington

candidate for the degree of Pharmacology *

Committee Chair

Dr. Ruth Keri, Ph.D

Committee Member and Thesis Advisor

Dr. Goutham Narla, M.D., Ph.D

Committee Member

Dr. Amar Desai Ph.D

Committee Member

Dr. Marvin Nieman

Committee Member

Dr. David Wald M.D., Ph.D

Date of Defense

January 17, 2020

*We also certify that written approval has been obtained for any proprietary material contained therein.

Dedication

“To laugh often and much; to win the respect of intelligent people and the affection of children; to earn the appreciation of honest critics and endure the betrayal of false friends; to appreciate beauty; to find the beauty in others; to leave the world a bit better whether by a healthy child, a garden patch, or a redeemed social condition; to know that one life has breathed easier because you lived here. This is to have succeeded.”

- Ralph Waldo Emerson

This work is primarily dedicated to my mother who raised me with the somewhat disillusioned mother’s belief that I have more talent in my pinky than others have in their whole body and, more importantly, that I have a lot to give back to others and share with the world. It is in her memory that I found my way to biomedical research and she is smiling ear to ear watching my success wherever she is now. This work is also dedicated to my father, who persistently taught me that said talent was useless without hard work. Lastly, this work is dedicated to my husband Angus whose love has buoyed me in a wild sea.

Table of Contents

List of Tables 5

List of Figures 6

Acknowledgments 8

Abstract 10

Chapter 1: Introduction and Background 12

1.1 Introduction to Phosphatases 13

1.2 Structure and stoichiometry of the PP2A Holoenzyme 13

1.2.1 Structural contributions to PP2A activity 14

1.2.2 Subunit stoichiometry and activity 16

1.3 Dysregulation of PP2A in human disease 16

1.4 Mechanisms of PP2A inactivation in cancer 18

1.4.1 PP2A subunits and cancer: Mutation, deletion, inactivation, 19

and aberrant expression

1.4.2 Post-translational modifications critical to PP2A activity 24

1.4.3. Endogenous inhibitors of PP2A 26

1.4.4 Exogenous inhibitors of PP2A 28

1.5 Approaches to activate PP2A in cancer 29

1.5.1 Inhibition of PP2A inhibitors 28

1.5.2 Promethylating agents 31

1.5.3 PME-1 inhibitors 31

1.5.4 Agents with undefined mechanism of action 32

1.5.5 Small Molecule Activators of PP2A 32

1.6 The oncogene MYC 35

1.6.1 MYC background 33

1.6.2 Regulation of the MYC gene 36

1.6.3 Structure and regulation of MYC protein 37

1.6.4 Approaches to target MYC at the transcriptional level 39

1.6.5 Inhibition of MYC protein and activity 40

1.7 Statement of Purpose 43

Chapter 2: Small molecule activation of PP2A for the treatment of MYC

driven cancers 55

2.1 Abstract 56

2.2 Introduction 56

2.3 Results 60

2.3.1 SMAPs inhibit tumor growth in c-MYC driven Burkitt’s Lymphoma 60

2.3.2 SMAPs decrease tumor burden and c-MYC expression in a KRAS model of

Non-Small Cell Lung Cancer 61

2.3.3 SMAPs inhibit tumor growth in c-MYC expressing xenograft

models of TNBC 63

2.3.4 SMAP treatment results in proteasome mediated MYC degradation 65

2.3.5 SMAPs inhibit the transcription of c-MYC target genes 67

2.4 Discussion 69

2.5 Experimental Procedures 71

2.6 Acknowledgements 77

Chapter 3: Summary of Discoveries and Future Directions 98

3.1 Summary 99

3.2 SMAPs for the management of N-MYC and L-MYC driven cancer 99

3.3 SMAPs, MYC and CIP2A 103

3.4 Understanding vulnerabilities in PP2A inactivated/MYC driven cancers 105

3.5 Status of the PP2A- B56a Holoenzyme 107

3.6 Understanding mechanisms of resistance to SMAPs in MYC driven cancers 107

References 111

List of Tables

Table 1.1 PP2A subunit alterations in cancer 48

Table 2.1: c-MYC target genes used to assess c-MYC transcriptional activity

and corresponding primer sequences for qRT-PCR 89

Table 2.2. Changes to c-MYC target genes in the Daudi cell line 90

Table 2.3 Changes to c-MYC target genes in the MDA-MB-231 cell line 91

List of Figures

Figure 1.1 Organization of the PP2A Holoenzyme 45

Figure 1.2 Structures of the PP2A Core Enzyme and Holoenzyme 46

Figure 1.3 PP2A mutations in cancer 47

Figure 1.4 Post Translational Modifications of PP2A 49

Figure 1.5 Structure of PP2A in complex with PME-1 and LCMT 50

Figure 1.6 Approaches to activate PP2A 51

Figure 1.7 Mechanisms of c-MYC regulation exploited in cancer and

approaches to target its expression and activity 52

Figure 1.8 Regulation of MYC protein 54

Figure 2.1 Structure of SMAP 1 and 2 79

Figure 2.2 SMAPs inhibit tumor growth and decrease c-MYC expression

in a model of Burkitt’s Lymphoma. 80

Figure 2.3 Figure 2.4 SMAPs inhibit tumor growth and decrease c-MYC

expression in KRAS driven NSCLC 81

Figure 2.4 SMAP treatment increases TUNEL staining and decrease

c-MYC protein expression in NSCLC mouse models upon

treatment with SMAPs 83

Figure 2.5 SMAPs inhibit tumor growth in models of triple negative

breast cancer 84

Figure 2.6 SMAPs decrease c-MYC expression through a proteasome

mediated mechanism and induce changes to c-MYC target genes 86

Figure 2.7 SMAPs do not induce changes to c-MYC mRNA in

Burkitts or breast cancer cell lines and inhibit c-MYC protein

in breast cancer cell 88

Figure 2.8 Confirmation of MYC overexpression in Daudi cell line 92

Figure 2.9. SMAP inhibition of tumor growth and changes to c-MYC

expression is abrogated by mutation to c-MYC phosphodegron 93

Figure 2.10 SMAP inhibition of tumor growth and changes to c-MYC

expression is abrogated by mutation to c-MYC phosphodegron 95

Figure 2.11 Confirmation of c-MYC band and overexpression in Daudi

tumor lysates 97

Acknowledgements

10 years ago I was a year into a post- baccalaureate pre-medical program, inspired to study medicine after taking care of my mom when she was sick and the interactions I had with her team of doctors. Yet, despite knowing I was on the right path, I was not sure a path through medical school was the way for me to find my way in this field. Enter Dr.

Goutham Narla. Literally. He entered the restaurant where I was working while supporting myself in school and our encounter lead to an interview which lead to a summer internship and eventually a full time research assistant position in his lab at

Mount Sinai Hospital in New York City. At the time I started in his lab I had barely finished a college level biology course, but I was hooked within a few weeks. A few years later I was applying to PhD programs. I interviewed at a handful of programs, well aware of the dogma to branch out and do my PhD with someone else, but all those experience did was tell me to trust my gut and do my PhD with Dr. Narla. Dr. Narla broadened my horizons and showed me what a career in biomedical research looked like.

He taught me how to think critically, to think big picture while focusing on the small details, and perhaps most importantly,he demonstrated each day a passion for his work that I hope to embody in my career- essentially to never lose sight of why we do what we do. Thank you Goutham.

I’d also like to thank my thesis committee for being supportive, providing insight and constructive criticism and teaching me how to receive feedback and think more deeply. Every meeting helped me become a better scientist and my work is better for it.

I’d also like to thank both Goutham and my thesis committee, Dr. Keri, Dr.

Nieman, and Dr. Wald and the pharmacology department for their patience and support.

My cancer diagnosis mid PhD truly disrupted my progress and my morale. In the months after chemo I was ready to give up multiple times on finishing my degree. But it was the support of these four that pushed me to persevere and it was their patience and understanding that allowed that to happen as I creeped into my 7th year of my PhD.

I have lived away from home since I was 16. As a result, I have developed a rich group of friends that have become my chosen family. Francesca, Amanda, Caite, Regina,

Tracey, Erin and Katie to name a few- strong women with incredible careers that have inspired and motivated me and didn’t think I was nuts when I decided to go back to school in my thirties. Your support got me here and helped me to the end. Thank you.

I left home at 16 because my family supported my dreams. At the time those were dreams of becoming a ballerina. But their unwavering support never waned as I found my way to a new career. Even when I was jumping from career to career in my twenties and not sticking to anything. Thank you for your encouragement – because of you I finally landed on my feet outside the dance studio when I could no longer land on them in the studio. Dad, Kathy- a special thank you for showing up in all the places mom would have. She must be so proud of the woman you’ve helped me become in her absence.

And a special thank you to my husband. You’ve never wanted anything for me but to chase my passions. You’ve seen me through grief, through cancer, and through a

PhD. I wouldn’t be surprised if the hardest of the three for you was getting through the

PhD. Having you by my side for all of these experiences taught me that life is truly better and easier with a best friend by your side; that it’s easier to ask for help than to insist on doing something yourself. Thank you and I love you.

“Hope the voyage is a long one” – C.P. Cavafy

Targeted Degradation of the MYC Oncogene Using Pp2a-B56a Selective Small

Molecule Modulators of Protein Phosphatase 2a as a Therapeutic Strategy for Treating

MYC-Driven Cancers

Abstract

CAROLINE C. FARRINGTON

A governing principle of cancer development is defined by a coordinate gain of oncogenic function and loss of tumor suppressor activity. To fully reverse this pathogenic process, one would want to simultaneously inhibit oncogene activity while reengaging tumor suppressor function. However, the majority of targeted therapies are directed at modulating the oncogenic gain with few therapies directed at the critical tumor suppressor proteins. This is based upon the dogma that, in a cell, it is easier to turn something off than to turn something back on. Indeed, activation of tumor suppressors using pharmaceutically tractable approaches have proven to be challenging. Yet, efforts persist to develop activators of tumor suppressor proteins. One that stands out as a therapeutic target is Protein

Phosphatase 2A (PP2A). PP2A is a serine/threonine phosphatase involved in the regulation of many cellular processes and is genetically altered or functionally inactivated in many cancers highlighting its central role in cancer pathogenesis.

One of the best-defined substrates of PP2A is the transcription factor c-MYC

(MYC). MYC, a well-described oncogene, is activated through both genetic amplification and stabilizing post-translational modifications. Cancers associated with high MYC expression are generally more aggressive. However, MYC has remained an elusive drug target as it lacks targetable drug pockets. MYC is rapidly degraded and its activity is inhibited by active PP2A. Thus, PP2A reactivation is a proposed strategy for the treatment of MYC driven cancers. Small Molecule Activators of PP2A (SMAPs) have been recently described for their potent anti-cancer activity which is dependent upon their ability to activate PP2A, reengaging its tumor suppressor activity.

This research demonstrates that activation of PP2A by SMAPs leads to MYC degradation resulting in the inhibition of cancer growth in both cellular and in vivo model systems. Biochemical and genetic tools are used to demonstrate that this anti-cancer activity is directly related to the degradation of MYC through the modulation of PP2A activity. In summary, the activation of tumor suppressor PP2A leads to the inhibition of

MYC, and establishes that SMAP mediated activation of PP2A is a novel approach that could be used for the treatment of MYC driven cancers.

Chapter 1: Introduction and background

Portions of this chapter were previously published in:

Farrington, C.C. *, Kastrinsky, D.*, McClinch, K. *Sangodkar J* and Narla, G

All Roads Lead to PP2A: Exploiting the therapeutic potential of this Phosphatase.

FEBS J, 2016, March; 283 (6), 1004-24. Review.

ÓJohn Wiley and Sons

1.1. Introduction to phosphatases

Complex processes in cell signaling require a set of molecular tools to modulate the activity and localization of specific proteins. Many of these responsibilities are regulated by kinases and phosphatases, whose opposite actions reversibly phosphorylate proteins. Due to their involvement in the progression of many cancers, the study of kinases has become an important field and one that has drawn considerable attention from the pharmaceutical industry over the past decade and a half. Despite their abundance and variety of substrates, the 518 human kinases display a high degree of similarity, and most features of their structure are conserved. In contrast, phosphatases exhibit considerably more structural variety with only a few enzymes performing the majority of the work.

Recent studies solidify their importance and the potential benefit of modulating their activity pharmacologically, but also reveal the inherent difficulties in designing tools to improve their function.

There are multiple families of phosphatases with diverse active sites and mechanisms. The major classes of phosphatases are protein tyrosine phosphatases (PTPs) and protein serine/threonine phosphatases (PSPs). The protein serine/threonine phosphatases consist of three families: phospho-protein phosphatases (PPPs), metal- dependent protein phosphatases (PPM), and DxDxT phosphatases (1,2). PPP, the phosphoprotein phosphatase family, is the largest containing several members including

PP1, PP2A, PP2B, and PP4.

1.2 Structure and stoichiometry of the PP2A holoenzyme

PP2A is a heterotrimeric complex. It consists of a scaffolding subunit (A), a regulatory subunit (B), and a catalytic subunit (C) (Figure 1.1). The A and C subunits each

exist with two possible variants α and b, with Aα and Cα accounting for the majority of each subunit in most cells (3-5). The four classes of the B subunit are: B (B55/PR55), B¢

(B56/PR61), B¢¢(PR48/PR72/PR130), and B¢¢¢(PR93/PR110)/Striatin. Each class contains

2-5 isoforms and additional splice variants. This predicts over 80 distinct combinations of the PP2A holoenzyme. This multitude of forms regulates PP2A’s activity and cellular localization and imparts specificity towards different substrates.

1.2.1 Structural contributions to PP2A activity

The PP2A A subunit is composed of 15 tandem HEAT repeats (Figure 1.1). HEAT repeats are named for the set of four cytoplasmic proteins first recognized to contain them

(Huntingtin, EF3, PP2A A subunit, and TOR1). Repeats contain approximately 40 amino acid residues organized into two anti-parallel α-helices. The helices are hydrophobic in nature enforcing their mutual attraction. In PP2A, the combination of these repeats forms a characteristic crescent structure (6). The C subunit binds to HEAT repeats 11-15 of the

A subunit (7). The C subunit embodies a globular structure with an α/b fold. This structure is homologous to other PPP catalytic subunits, however, the subunits are not interchangeable among the different enzyme types (8). The molecular basis for these interactions was described when the AC dimer, also known as the “Core Enzyme” crystal structure was solved (9). The C subunit active site contains 2 manganese atoms that bind to phosphate and facilitate the hydrolysis of serine/threonine phosphate esters. The active site is positioned away from the ridge of the A subunit HEAT repeats and proximal to the site where the B subunits bind.

The core enzyme interacts differently with each class of B subunit (Figure 1.2A).

Crystal structures have been solved for the B, B¢, B¢¢ family of subunits while less is known about the B¢¢¢ family. The B/PR55 family contacts the scaffolding subunit via two extended interfaces (Figure 1.2B). The first is a seven-bladed propeller, each composed of a WD40 repeat. The second is a b-hairpin handle with additional secondary structures. The bottom face of the propeller binds to the HEAT Domains 3-7 of the A subunit, and the b-hairpin handle interacts with HEAT repeats 1 and 2. The top face of the propeller is the proposed substrate binding site. While proximal to it, the B/PR55 subunit makes very few contacts with the C subunit (10-12). The structure of the B¢/PR61 family of subunits is strikingly similar to the A subunit, containing 8 HEAT-like repeats (Figure 1.2C). These interact with the A subunit at HEAT repeats 2-8 and also interact with the C subunit. The B/PR55 and

B¢/PR61 subunits bind similarly to the core enzyme such that the substrate binding site lies on the top face of the B subunit proximal to the C subunit active site. The proximity of these B subunits to the active site explains their role in conferring specificity for substrate proteins (9,13).

The structures of the holoenzyme with B¢¢ subunits (PR70/PR72) were solved recently (Figure 1.2D). The B¢¢ family consists of linear arrangements of different functional motifs with a substrate binding region near the C-terminus. This arrangement differs from the B and B¢ whose substrate binding sites are found on their top surfaces and involve their structural repeats. The B¢¢ family also includes an N-terminal hydrophobic motif with two EF-hand calcium-binding motifs that bind to the A subunit at HEAT repeats

1-7. The subunit also contacts the C subunit near the active site via a helix (439-446) positioning the substrate binding site next to the active site (14).

The A subunit orchestrates the formation of the active holoenzyme through its conformational flexibility. Binding with the C subunit shifts HEAT repeats 13-15 by 20-

30 Å, while binding with the B¢ subunit forces an N-terminal repeat to twist up to 50-60 Å and rearranges the hydrophobic core within A (9,13). For the B¢¢ family, the A subunit adopts a compact conformation relative to other holoenzymes, reducing its width and increasing its height. An additional helix domain of PR70 extends beyond the A subunit making it wider than the other holoenzymes (14).

1.2.2 Subunit stoichiometry and activity

PP2A constitutes approximately 1% of total cellular protein. The nature of its production, assembly, and subunit stoichiometry is still unclear. To address these questions, studies were conducted in yeast, which contain a smaller repertoire of subunit variants. The ratio of subunits (A:B:C) in yeast was originally determined to be 1:4:8, suggesting that production of A is a limiting factor (15,16). However, another study looking at global protein expression suggested the ratio was quite different; the ratio of subunits A:B:C instead being 17:9:10 (16,17). In mammalian cells, the A subunit is expressed in excess of the other subunits (18). There is consensus that the monomeric C subunit is unstable and requires binding to the A subunit or other non-canonical B subunits to preserve its activity (19-21). Moreover, binding to the A subunit may enhance the stability of the B subunits and fulfill other housekeeping roles (21-23).

1.3 Dysregulation of PP2A in diseases

PP2A related pathways are perturbed in many diseases. In both cancer and neurodegeneration, the common pathological mechanism involves activated kinase signaling pathways combined with the loss of PP2A activity. In the case of neurodegeneration, PP2A dysfunction leads to increases in hyperphosphorylated tau. Tau protein normally stabilizes microtubules. Hyperphosphorylated tau is thought to play an important role in the etiology of Alzheimer’s disease by forming neurofibrillary tangles.

The PP2A Bα isoform is the primary tau binding phosphatase. PP2A mediates ∼71% of total tau phosphatase activity in the human brain (24) and dephosphorylates abnormally phosphorylated tau at Ser46, Ser199, Ser202, Ser396, and Ser404 (25). Alterations in PP2A regulating proteins, its catalytic activity, subunit expression, methylation, and phosphorylation patterns have been reported in Alzheimer's disease-affected brain regions.

For example, reduced PP2A mRNA (26), protein levels (27), and phosphatase activity

(25,28) were observed in the brains of Alzheimer’s patients. Increased phosphorylation of

PP2A at Tyr307 has been found in phospho-tau-rich, tangle-bearing neurons (29). Attempts to reduce this phosphorylation through kinase inhibition generated limited success clinically. Consequently, there is a growing interest in developing PP2A-targeted therapies for Alzheimer's disease. These include disruption of inhibitory protein-protein interactions, modulation of disease relevant post-translational modifications, and allosteric activation

(30,31).

In contrast, PP2A performs the opposite role in diabetes. Where these pathways are functioning normally, glucose homeostasis requires the insulin-mediated activation of

PI3K-AKT and downregulation of PP2A (32). This leads to stimulation of the GLUT4 transporter resulting in uptake of glucose into skeletal muscle tissues. This decreased PP2A

activity is absent in skeletal muscle tissues of individuals with type II diabetes and leads to impaired insulin sensitivity (33). A plant derived natural product, carnosic acid, also discussed below, stimulates proper glucose metabolism by activation of AKT via C subunit demethylation (34). Therefore, PP2A deactivation might be a useful target for ameliorating dysregulated glucose metabolism that is characteristic of type II diabetes.

There are additional reports that implicate a positive role for PP2A in inflammatory lung diseases like asthma and COPD (35) and in heart function (36). These effects are attributed to PP2A’s inhibitory effects on the mediators of inflammation, and on a variety of substrates involved in cardiac muscle contraction, respectively. Given that cancer,

Alzheimer’s disease, diabetes, asthma, and COPD claim millions of lives each year and cost billions to treat in the United States alone, developing drugs that modulate relevant targets such as PP2A in these indications is an attractive option.

1.4 Mechanisms of PP2A inactivation in cancer

PP2A’s essential role in homeostasis can be inferred from the increasing number of disease states in which it is functionally inactivated. Numerous studies highlight the role of PP2A as a tumor suppressor and suggest that disruption of the PP2A holoenzyme may contribute to the development of cancer. In cancer, PP2A is inactivated through several mechanisms including: somatic mutation, phosphorylation and/or methylation of the C terminal tail of the catalytic subunit (Figure 1.3) and through increased expression of endogenous PP2A inhibitors (Figure 1.6) (37). Several of the genetic alterations prevent the A subunit from binding to the B and/or C subunits, resulting in disruption of the core enzyme and complex (5,38-40). Understanding these defects will enhance the future

development of PP2A targeted therapeutics by facilitating the selection of the correct patient cohorts and suggesting effective combination therapies.

1.4.1 PP2A subunits and cancer: Mutation, deletion, inactivation, and aberrant expression

Scaffolding subunit: PP2A Aα and Ab

Mutations have been detected in all subunits of PP2A in cancer, but the gene encoding the Aα subunit, PPP2R1A, has the highest mutation rate. PP2A is an essential enzyme, therefore cancer-associated PP2A Aα mutations in clinical specimens typically involve only a single allele (23,41). These mutations create a state of haploinsufficiency.

Point mutations most commonly occur in the Aα subunit and ~30% of these mutations occur at a mutational hotspot within HEAT repeat 5. While the functional relevance of some of the identified Aα mutations have been studied, the significance of the mutations in this hot spot region has yet to be elucidated (39,42). Most reported PP2A Aα mutants are unable to bind the regulatory subunits, and, in particular, to members of the B¢ family

(B56) (38). For example, when R418W and D171–589, two point mutations in Aα that were first detected in melanoma and breast carcinomas respectively, were studied, these mutations led to reduced binding to the C subunit and to all of the B subunits tested. Two additional mutants, E64D and E64G, specifically lost efficient binding to the B56 family subunits (5,39) and resulted in a state of haploinsufficiency in transgenic mice (23). In addition, mice containing the E64D mutation showed an increased incidence of lung cancer when exposed to benzopyrene and decreased survival when crossed with KRASG12D mice

(39). To date, mutations in PPP2R1A have been identified in breast, lung, melanoma,

ovarian, endometrial, uterine, and colon cancers (41,43-47) and decreased expression of

Aα was detected in human gliomas (48). (Table 1)

Akin to PP2A Aα mutations, cancer-associated Ab mutations also induce haploinsufficiency and impaired binding to the B and C subunits (38-40). The gene that encodes the β isoform of the A subunit, PPP2R1B, is located on a chromosomal band frequently deleted in cancer cells, 11q23 (49,50). According to a seminal paper by Wang et al., which was the first to demonstrate PPP2R1B to be mutated in human cancers, the

11q23 gene locus displayed loss of heterozygosity in 30-50% of breast, lung, ovary, cervical carcinomas, melanomas, and in 15% of non-Hodgkin's lymphomas (NHL) and chronic lymphocytic leukemias (CLL) (51). Many of the mutations in PPP2R1B are missense mutations including G8R, P65S, G90D, L101P, K343E, D504G, V545A, V448A.

One contains the double mutant L101P/V448A and one contains an in-frame deletion

DE344–E388 (40,52). The DE344–E388 mutant was found to be incapable of binding to any of the B subunits tested (40,52). One mechanism of inactivation unique to the b isoform of the scaffolding subunit is abnormal RNA splicing leading to aberrant transcripts of PPP2R1B. Alternative splice variants of PP2A Ab were observed in B-cell chronic lymphocytic leukemia (B-CLL). These aberrant transcripts were incapable of binding B and C subunits, which subsequently led to a loss of PP2A activity (53). In addition, 29% of hepatocellular carcinoma (HCC) tumors and 3% of corresponding non-tumor tissues tested showed co-expression of wild-type and aberrant mRNA of PPP2R1B, suggesting that alternative splicing may facilitate the development of HCC (54).

Analogous to AKT, the GTPase RalA participates in transcription, migration,

transport, apoptosis, and cell proliferation. PP2A Aβ binds and regulates the activity of

RalA by dephosphorylating RalA at Ser183 and Ser194. This dephosphorylation leads to inactivation of RalA, again highlighting that PP2A Aβ functions as a tumor suppressor.

Mutations in Aβ disrupt this interaction leading to constitutive activation of RalA that results in transformation (52).

In summary, extensive sequencing of human samples and cancer cell lines has revealed PPP2R1B to be mutated in many solid cancers including breast, lung, colon, melanoma, ovarian, cervical, HCC, NHL, CLL, and B-CLL. Although it is 40 times less abundant than Aα (55), PP2A Ab clearly has a role in the tumor suppressor capabilities of

PP2A as mutations and loss of expression of the PPP2R1B gene inhibit this activity. There has been some speculation that PP2A Aα can compensate for Ab mutation and or loss of expression in cancer. However, several reports suggest that this is unlikely. While Aα and

Ab are 85% identical, they have vastly different B and C subunit affinities and have distinct biochemical properties (13,40). For instance, Ab mutations show decreased binding to the

B and some of the B¢ regulatory family members but predominantly affect processes involving the catalytic subunit and regulatory PR72 -containing holoenzyme. In contrast,

Aα subunit mutations mostly affect pathways where B¢-containing holoenzymes are instrumental (5,38,40). In addition to this, complexes involving Ab and regulatory subunits regulate the phosphorylation of specific substrates involved in cellular transformation that are distinct from pathways regulated by the Aα-regulatory subunit complexes (38). Lastly, in experiments in transgenic mice, overexpression of Aα could not revert tumorigenesis that was induced by suppression of Ab (5). Together, this

evidence strongly suggests that PP2A Aα and Ab are functionally different and cannot compensate for each other.

Regulatory subunits: PP2A B, B¢, B¢¢, and B¢¢¢

Mutations in the regulatory subunits of PP2A occur at much lower frequencies than those in the A subunit and most commonly result in decreased expression of the B subunits.

Other methods of inactivation include deletion, DNA hypermethylation, and one point mutation found in lung cancer. The F395C mutation detected in lung cancer occurs in a region necessary for PP2A-B56g-p53 interaction. In cell culture based studies, the B56g mutant protein was unable to interact with p53, thereby inhibiting its p53 dependent tumor suppressive functions (56). A study of 141 prostate cancer samples using an Affymetrix

SNP array found that PPP2R2A, which encodes the regulatory subunit, B55α, was deleted in 67.1% of the tumor samples tested. Moreover, homozygous deletions occurred in three of the prostate cancer samples (57). Deletions in the PPP2R2A gene have also been reported in breast cancer and myeloma (58,59).

In addition to mutations, loss of B subunit protein expression has been linked to cancer progression. Decreased expression of both PPP2R5A and PPP2R5C, which encode

B56a and B56g, respectively, were reported in melanoma, with the lowest levels of expression in metastatic tissues (60-62). In addition, immunohistochemical analysis of

PPP2R2C protein levels in primary prostate tumors determined that loss of PPP2R2C, which encodes B55g, was highly correlated with metastasis and prostate cancer specific mortality (PCSM) (63). Furthermore, in a cohort of 231 patients with acute AML, B55α expression was inhibited and was associated with increased AKT phosphorylation at

threonine 308 and loss of complete hematological remission. B55α dephosphorylates AKT at T308 and when suppressed, it leads to constitutive activation of AKT and enhanced proliferation. In a study by Ruvolo et al., remission duration was evaluated in 231 newly diagnosed AML patients evaluated at the MD Anderson Cancer Center. Patients were divided into two groups based on their B55a protein expression level: B55a-high and

B55a-low. Kaplan–Meier survival curves illustrated the effect of B55α expression level on remission duration. Patients in the B55α-low group experienced significantly shorter complete remission duration than those in the B55α-high group (64). The identification of

B55α as a specific regulator of AKT phosphorylation at Thr308 as well as B55α expression’s correlation with remission duration highlight its potential to serve as a biomarker in AML.

Lastly, epigenetic alterations are a method of PP2A inactivation that is unique to the regulatory subunits. PPP2R2B, which encodes B55β, may be inactivated through epigenetic silencing according to a study by Muggerud et al., which detected an increase in DNA methylation of the PPP2R2B gene in ductal carcinoma in situ and locally advanced breast tumors (65). Furthermore, B55β is epigenetically inactivated by DNA hypermethylation in colorectal cancer (CRC). This inactivation was shown to effect MYC signaling. A study by Tan et al. demonstrated that epigenetic inactivation of PPP2R2B occurred in >90% of patient derived CRC tumor samples tested. They found that loss of

PPP2R2B expression led to the induction of PDK-1 dependent MYC phosphorylation at serine 62 by the mTOR inhibitor, rapamycin, which subsequently led to resistance.

Restoration of PPP2R2B expression abrogated MYC phosphorylation, resensitizing CRC cells to rapamycin. As clinical responses to rapamycin are quite variable, better biomarkers

are needed to predict which patients are most likely to respond to treatment. Tan’s study highlighted the potential of PPP2R2B to act as such a biomarker for selecting patients who may respond best to rapamycin treatment (66). In summary, given the critical role the regulatory subunits play in determining the substrate specificity of the PP2A heterotrimeric complex, identifying mutations in these subunits and understanding their functional implications remains an active area of research.

Catalytic subunit: PP2A Cα and Cb

To date, the only reports of mutations in the C subunit have been in prostate cancer and AML. A genome wide expression study identified significant downregulation of

PPP2CA, which encodes the α isoform of the catalytic subunit, in androgen insensitive prostate cancer cell lines compared with androgen sensitive lines (67). This finding was subsequently confirmed at the protein level and in human clinical samples (68). In addition, PPP2CA was found to be downregulated in a cohort of patients with P53 mutant

AML(69,70). There have been no reports to date of mutations in PPP2CB, the gene that encodes the b isoform of the catalytic subunit.

1.4.2 Post-translational modifications critical to PP2A activity

Methylation at L309 on the C subunit C-terminal tail

The C subunit C-terminus undergoes methylation at L309 (Figure 1.4A and 1.4B).

This modification is regulated by two enzymes: LCMT-1, an S-adenosylmethionine

(SAM)-dependent methyltransferase that is expressed in the cytoplasm and PME-1 (Figure

1.5A), a lipase-like methylesterase, that is expressed in the nucleus. While LCMT-1 mediated activation of PP2A is reversible (Figure 1.5B), PME-1 mediated demethylation

is not truly reversible because it denatures the active site (71-73). The role of this methyl modification is complicated, and both enzymes are essential (74,75), and likely exhibit control on cell cycle and development (76). Decreased methylation typically corresponds with increases in cancer progression because methylation enhances holoenzyme assembly, specifically of the preformed AC core enzyme with B subunits. L309 methylation is not essential for every variant (PR61/B¢, PR72/B¢¢), but is essential for some (PR55/B)

(2,10,77-82).

The role of L309 methylation gained in popularity in part due to its putative role in neurodegeneration. PP2A/PR55α is a predominant brain expressed phosphatase that requires L309 methylation for holoenzyme formation. Folate is a key nutrient in the production of the LCMT-1 cofactor, S-adenosylmethionine (SAM), which supplies the enzyme’s methyl donor. In folate deprived neuroblastoma cells, both PR55α and LCMT-1 expression levels are diminished, and PP2A exists in demethylated forms. This state leads to tau hyperphosphorylation and cell death, a signature of neurodegenerative diseases (83).

In mouse models of Alzheimer’s, levels of LCMT-1, methylated C subunit, and PR55α are decreased. Restoration of methylation by overexpression of LCMT-1, and induced expression of PR55α restores neurite outgrowth, a signature of disease remission (84).

Methylated PP2A is typically found associated with unphosphorylated tau, localized to the plasma membrane. In contrast, demethylated PP2A is improperly localized and not associated with tau (85). An herb-derived compound, cornel iridoid glycoside, reverses tau phosphorylation by inhibiting C subunit demethylation (86). The development of natural products and small molecules to modulate tau hyperphosphorylation would provide much needed tools and therapeutics for Alzheimer’s disease.

There is also evidence that PME-1 inhibits PP2A independently of its role on C subunit demethylation. Association with PME-1 possibly stabilizes PP2A in an inactive conformation and creates a cellular pool of enzyme that can be activated when required.

This association is regulated by the interplay of PP2A with another protein, Phosphatase

Activator (PTPA) which activates the PME-1 bound form of PP2A (Figure 1.5C) (87).

PTPA is a protein with an elusive functional role; however, it is commonly associated with

PP2A and possesses a chaperone-like function for the correctly folded C subunit (88). It also possesses a peptidyl-prolyl cis ⁄ trans isomerase activity that acts on Pro190 of the C subunit, inducing a conformational change that may contribute to the reactivation of PME-

1 bound PP2A (89,90). Additionally, like PME-1 and PTPA, several other proteins associate with the PP2A core or trimer that are not classified as B subunits. The α4 protein is a PP2A binding protein that stabilizes PP2A and other PPP enzymes by binding to the C subunit and preventing its degradation (91,92). The α4 protein is essential and plays a role in cellular adaptation to stress by preserving stocks of PP2A. This PP2A store ultimately dephosphorylates the accumulated products of stress responses (17).

1.4.3 Endogenous inhibitors of PP2A

PP2A is commonly inactivated in cancer by the overexpression of its endogenous inhibitors. The most prominent deactivation mechanism, this occurs in up to 90% of cases in lung and breast cancers and is often associated with poor response to current therapies.

There are several endogenous inhibitory proteins that inactivate PP2A (Figure 1.6).

Inhibitor 1 of PP2A (I1PP2A), also known as ANP32A, inhibits PP2A activity in human umbilical vein endothelial cells (93,94). I1PP2A also binds to sphingosine, and this

interaction abrogates its binding to PP2A resulting in PP2A activation (95). Additionally, the greatwall kinase (Gwl) might function as an inhibitor of PP2A. Gwl activates ENSA and Arpp19, which are phosphorylation-dependent inhibitors of PP2A (96,97). GWL mediated inhibition of PP2A-B55 leads to phosphorylation of Cdk1 substrates and mitotic entry. Furthermore studies have shown that HOX11, a homeobox gene rearranged in T- cell leukemia by chromosomal translocation, inhibits PP2A (98). However, the two endogenous inhibitors of PP2A most overexpressed in human cancers and best characterized are SET and CIP2A (94,99). SET, also known as inhibitor-2 of PP2A

(I2PP2A), binds to the C subunit of PP2A. It was discovered as a chimeric protein in a patient with acute undifferentiated leukemia. In this case, SET was a translocated gene fused with nucleoporin (CAN gene) (100-102). SET displays increased expression or increased activity in several cancers such as CML, AML, and B-cell CLL, colorectal cancer, breast cancer, and lung cancer (103-109). In addition to its overexpression, altered phosphorylation of SET also inactivates PP2A (110,111). Studies in Alzheimer’s disease have elucidated that Val92 at the amino-terminal fragment and the amino acids 176-277 on the C-terminal region of SET are important for PP2A binding. Furthermore, accumulation of SET in the cytoplasm is regulated by phosphorylation of Ser9 in the nuclear localization signal (112-114).

CIP2A (Cancerous Inhibitor of PP2A) is a PP2A interacting protein. CIP2A is most strongly associated with inhibiting the activity of PP2A on c-MYC resulting in c-MYC stabilization and consequential proliferation. Inhibition of PP2A by CIP2A is also associated with the stabilization of other pro-survival and pro-growth proteins including

E2F1, mTOR, and DAPk, resulting in the inhibition of senescence, autophagy and

apoptotic pathways respectively (115-117). Conversely, the depletion of CIP2A results in a decrease in cancer cell viability. It is not understood how CIP2A inhibits PP2A but some reports suggest that it interacts with the A subunit and perhaps the C subunit, preventing the interaction of the active site with target proteins (118). Encoded by the KIAA1524 gene,

CIP2A is overexpressed and may be prognostic in lung cancer, breast cancer, pancreatic cancer, bladder cancer, osteosarcoma, esophageal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, hepatocellular carcinoma, and colorectal cancer (119-

136). This abundant clinical relevance makes CIP2A an important therapeutic target.

1.4.4 Exogenous inhibitors of PP2A

There are a number of microbial, marine, and insect derived natural products that bind and inhibit PP2A and other PPP members. These include okadaic acid, fostriecin, microcystins, calyculins, cantharidin, and dragmacidins. Many were isolated from screens of natural product extracts for cytotoxins. Others were discovered in pulldown studies of biologically active extracts, as PP2A binding small molecules (137). While it seems counterintuitive to inhibit a tumor suppressor like PP2A, at the time, the potency of these compounds in cytotoxicity assays generated much interest in their potential clinical uses for cancer. Their toxicity underscores PP2A’s essential role in regulation, and several of these compounds provided extremely useful tools for exploring PP2A’s functions. The natural product toxins bind into or adjacent to and obstruct the C subunit active site. (9,138)

The same could be said for the Simian Virus 40 (SV40) small t antigens (ST). SV40 ST consists of an N-terminal J domain and a C-terminal unique domain that contains two separate zinc-binding motifs. SV40 ST interacts with the core enzyme by binding to the

B56 subunit binding site on PP2A Aα (HEAT repeats 3-7), causing displacement of the B subunits (139). This displacement perturbs the function of PP2A and its activity towards multiple substrates (140,141). While not directly tumorigenic in humans, these viruses transform cells and can promote tumor growth.

1.5 Approaches to activate PP2A in cancer

1.5.1 Inhibition of PP2A inhibitors

SET

The association of SET with cancer inspired several attempts to target this inhibitor for PP2A activation. One strategy to inhibit SET involves ApoE (apolipoprotein E), a multifunctional holoprotein with a role in cholesterol transport (142-144) and immunoregulatory functions (145-147). ApoE and apoE-mimetic peptides, COG112 and

COG449 (OP449), bind to SET resulting in activation of PP2A (148-151). SET antagonism with OP449 results in cytotoxic activity with demonstrable efficacy in the treatment of

CML and AML (148).

FTY720 (Fingolimod/GilenyaÒ), originally approved for use in multiple sclerosis by

Novartis, activates PP2A via inhibition of SET. FTY720 was derived from a fungal metabolite (152,153) and acts as an immunosuppressant by modulating the sphingosine-1- phosphate (SIP) receptor (154-156). FTY720 exerts anti-tumor activity in breast, HCC, glioma, and multiple myeloma models. Specifically, in CML, activation of PP2A (105,157-

159) by FTY720 induces apoptosis through the inactivation of BCR-ABL1 and negative regulation of several survival factors including ERK. Finally, ceramide is a sphingolipid that activates PP2A in several cancers and induces apoptosis (160-164). Some reports

implicate a direct interaction of ceramide with PP2A. Others suggest that ceramide activates PP2A by inhibiting the interaction between PP2A and SET (165). Ceramide induces apoptosis in prostate cancer cells through PP2A mediated induction of p27 (162).

CIP2A

Several natural products possess activities that are relevant to PP2A activation via

CIP2A inhibition. Celastrol (tripterine) caused a proteasome-mediated degradation of

CIP2A resulting in inhibition of proliferation and induction of apoptosis in lung cancer

(166,167). Celastrol induced rapid degradation of CIP2A through the interaction of the E3 ligase, CHIP. In vivo studies showed that celastrol potentiated the effects of cisplatin suggesting that celastrol could have therapeutic implications in lung cancer.

Ethoxysanguinarine (ESG), a benzophenanthridine alkaloid, downregulates CIP2A resulting in an increase in PP2A activity and a consequential downregulation of c-MYC and AKT in lung cancer. The downregulation of CIP2A subsequently results in inhibition of proliferation and induction of apoptosis in lung cancer (168,169). Combined treatment with ESG enhanced the effects of cisplatin in lung cancer.

The anti-cancer drug bortezomib might provide another strategy for activating

PP2A via CIP2A inhibition. Bortezomib, a dipeptidyl boronic acid, is a proteasome inhibitor first approved for the treatment of multiple myeloma. It blocks the degradation of

IkB, an inhibitor of NF-kB (170,171). In subsequent studies, bortezomib negatively regulated transcription of CIP2A resulting in decreased AKT phosphorylation and induction of apoptosis in breast cancer (172). It enhances PP2A activity in HCC (173).

Finally, derivatives of erlotinib that were devoid of anti-EGFR activity, inhibited

production of CIP2A causing subsequent decreases in AKT phosphorylation and cell growth inhibition (174).

1.5.2 Promethylating agents

One strategy to activate PP2A could be the induction of methylation. As mentioned previously, PP2A methylation at residue Leu309 enhances the affinity of the PP2A core enzyme for the regulatory subunit (73,175-179). Some reports suggest that PP2A methylation is linked to PP2A activity and that PP2A methylation induced by an agonist, such as xylulose-5-phosphate, would cause an increase in PP2A activity (178,180,181).

This increase in PP2A activity results in a decrease in AKT and c-MYC expression and a decrease in proliferation (182). Furthermore, DNA damaging agents such as chloroethylnitrosourea (CENU), induce PP2A methylation, increasing PP2A activity resulting in inhibition of AKT and c-MYC (182,183).

1.5.3 PME-1 inhibitors

PME-1 garnered increasing attention due to its role in cancer and its association with PP2A (184). To develop probes for annotating enzymatic function, the Cravatt laboratory used its activity based protein profiling technique to screen for inhibitors of serine hydrolases. Their methods generated two independent small molecule inhibitors of

PME-1. The first, an azalactam (ABL127, IC50 = 10 nM) (185), and the second, a sulfonyl acrylonitrile covalent inhibitor derived from a complementary screening library (186).

These will hopefully provide useful leads to validate PME-1 as a drug target.

1.5.4 Agents with undefined mechanism of action

Several compounds activate PP2A by unknown mechanisms. Forskolin, derived from the root of Coleus forskohlii, is a diterpenoid natural product used for many conditions including cancer. By activating adenylate cyclase, forskolin increases intracellular concentration of cyclic adenosine monophosphate (cAMP) (187). Forskolin treatment reduces phosphorylation at Y307 on the C-terminal tail of PP2A C thereby activating PP2A (188-190). In AML, forskolin increased PP2A activity resulting in decreases in proliferation, induction of apoptosis, and changes in the phosphorylation of

AKT and ERK (188). Perphenazine and other phenothiazine containing tricyclic neuroleptics, activate PP2A in T-ALL cells through direct binding of the PP2A Aα subunit

(191).

Carnosic acid is a polyphenolic diterpene that activates PP2A in prostate cancer.

This activation results in negative regulation of the AKT/IKK/NF-kB pathway (192).

Vitamin E analogs, such as α-tocopheryl succinate, inhibit proliferation in several cancers

(193-203). α-Tocopheryl succinate promoted activation of PP2A inactivates JNK signaling

(204,205).

1.5.5 Small Molecule Activators of PP2A

In addition to the methods described above, in recent years, an approach to directly activate PP2A has emerged using a series of small molecules originally derived from a class of FDA- approved anti-psychotics. These molecules, phenothiazines that include the drugs Trifluoperazine and Chlopromazine were observed to have an anti-cancer effect.

Derivitization of these molecules revealed a potent new class of small molecules absent of

the dopamine activity in the parent molecules and with enhanced anti-cancer activity that was well tolerated in in vivo studies. Further investigation revealed that these molecules activate PP2A and have since been named Small Molecule Activators of PP2A (SMAPs).

SMAPs have been shown to have potent anti-cancer activity in a diverse set of cancers including prostate, pancreatic and non-small cell lung cancers, cancers driven by potent oncogenes like the Androgen Receptor and Ras. In these models, SMAPs inhibit cancer cell growth in vitro as well as in multiple in vivo models. Moreover, consistent with the hypothesis that anti-cancer activity is dependent on their interaction with PP2A, a series of chemical and genetic studies to inhibit PP2A or inhibit SMAP binding to PP2A abrogates the observed anti-cancer activity.

Research to date suggests that SMAPs bind to A scaffolding subunit of PP2A.

Specifically, a combination of radio radiolabeled equilibrium dialysis, photo cross-linking followed by tandem MS chromatography, and hydroxy-radical footprinting approaches confirm that SMAPs bind the PP2A-Aα subunit. By cryo-EM, the PP2A holoenzyme containing the regulatory subunit B56a along with SMAP DT-061 revealed a binding pocket on A where both the B and C subunits interacted as well. This data shows that the molecule interacts with E100 and E101 on the A subunit, K316 on the B subunit and F308 of the C subunit. While the C subunit anchors on the C-terminal region of A and the B subunit anchors on the N-terminal region of A, the tail of C subunit sandwiches itself between B and A subunits near where DT-061 binds. This tail has previously been shown to be inherently flexible, but the interaction of DT-061 and Y307 on the C-tail pins the tail near the A-B subunit binding site and it is believed that this stabilizes the holoenzyme and enhances activity of the A-B56a-C holoenzyme. Additionally, this study showed that DT-

061 binding to PP2A may select for a small subset of PP2A complexes within the B56 family. This is especially relevant as one of the best characterized substrates of the B56a containing holoenzyme is MYC, described in more detail below, a potent oncogene.

Lastly, and most important when discussing the use of this approach for the treatment of cancer in patients one day, studies of the pharmaceutic profile of SMAPs demonstrate a pharmaceutic profile that is promising for their potential development into a therapeutically tractable approach. In a 7 day safety study in mice, concentrations of

SMAPs up to 800mg/kg once daily were well tolerated. Specifically, throughout the study, there were no observable changes in body weight, food consumption and behavior. At the termination of the study, it was determined that there no changes in total protein (both albumin and globulin fractions), bilirubin, creatinine, glucose, cholesterol, ALT, AST and

SDH as assessed by a clinical chemistry profile. Additionally, histology did not reveal any microscopic findings in the liver. In an acute TK study, it was found that SMAPs are bioavailable with a plateau in absorption between 300 and 600 mg/kg and the Cmax and

AUC were comparable on the first day of exposure. At the end of 7 days, the area under the curve over 24 hours (AUCO-24) demonstrated that exposure remained high. Lastly, the calculated half-life of the molecule is 6.5 hours in vivo.

Longer efficacy studies in vivo demonstrate efficacy at concentrations of 5-15 mpg twice a day (BID) versus once a day. Additionally, short term studies to study the pharmacodynamics (PD) of the molecules revealed sustained target modulation for at least

12 hours. Combined, this safety profile along with the observed efficacy and target modulations suggests that activation of PP2A using this class of small molecules is a promising approach to study further.

1.6 The oncogene MYC

1.6.1 MYC background

The MYC oncogene encodes the c-MYC protein (MYC), a nuclear transcription factor. It belongs to a family of transcription factors that include c-MYC (MYC), L-MYC, and N-MYC. MYC was the first of the family to be discovered after studies into chicken tumors were revealed to be caused by a viral oncogene v-myc that is homologous to

MYC(206,207). v-myc was later shown to have co-opted itself from the host (chicken) DNA for myc.(208,209) Early observations that confirmed the oncogenic potential of MYC include the identification of a translocation in Burkitt's Lymphoma that renders MYC constitutively active driven off the promoter for IgH(210,211). This observation is the first to confirm its potential as an oncogene. Since then, additional translocations, amplifications, and the identification of signaling pathways that activate MYC have been mapped and further confirm its broad oncogenic potential and activity. In vitro and in vivo studies have confirmed its ability to transform normal cells and drive tumorigenesis. (212-

223). However, a common observation in most studies is that MYCs oncogenic potential is dependent on other genetic alterations to enable it’s transforming or tumorigenic potential. Settings include the addition of other oncogenes like KRAS as well as the suppression of tumor suppressor proteins like P53 or pertinent to this work, PP2A.

(213,215,222,224-228).

Of the three family members, MYC is the most commonly expressed and it is expressed broadly in different tissue types. (MYCN) N-MYC is fairly well characterized for its role in development as well as cancer and is expressed primarily in tissue of neuronal

origin. MYCL(L-MYC) is the least characterized of the family, identified in lung tissue but less studied for its function and role in cancer(229-231).

As a transcription factor, MYC regulates several gene expression programs associated with cell growth, proliferation, metabolism, apoptosis, and transformation(221,222). In non-cancerous cells its expression is tightly regulated but its aberrant overexpression and stabilization, described in more detail below drives tumorigenesis across a myriad of cancer types, from blood cancers to solid tumors.

Moreover, in settings of MYC overexpression or MYC stability, genetic silencing of MYC greatly inhibits cell growth and sensitizes cancer cells to current standards of care(222,232-

235).

Overall, research to date highlights the benefit of targeting MYC in cancer.

However, as a transcription factor, it is inherently more challenging to design direct inhibitors towards it. Transcription factors are notable for the highly disordered regions.

These regions enable transcription factors to bind high and low-affinity binding sites and a diverse set of co-factors but it also makes them very flexible and thus challenging in the context of traditional drug development strategies aimed at protein-protein interactions or

DNA binding.(236,237) However, these challenges have been met with many innovative strategies to target MYC with their own strengths and limitations.

1.6.2 Regulation of the MYC gene

The MYC gene is found on chromosome 8, contains three exons- exon 1 is a large non-coding exon, and exon 2 and 3 are coding exons, containing two major start codons-

CTG and ATG, and its transcription is driven by 4 distinct promoters (P0, P1, P2, P3) . The

majority (~75%) of the MYC transcripts are driven off the P2 promoter (220,238,239).

Overall though, MYC promoter control has been demonstrated to be very complex, regulated by a number of signaling pathways, other transcription factors, cis-regulatory elements, chromatin remodeling as well as auto suppression. (238,239). These regulatory events can converge in a myriad of combinations in response to various signals to activate the MYC promoter and regulate transcription. But in cancer cells, a number of these regulatory events can be dysregulated or exploited that lead to MYC amplification. As mentioned previously, MYCs oncogenic potential was first confirmed when it was identified as the driver of Burkitt's lymphoma. Its constitutive expression in this disease is the result of a chromosomal translocation that results in MYC being driven off the promoter for the immunoglobulin heavy chain (IgH). Since then additional translocations have been identified that drive MYC expression. However, more frequently, high MYC expression appears to be the result of aberrant genomic changes such as gene amplification- small focal amplifications and large amplifications, epigenetic mechanisms (histone acetylation), activated super-enhancers, and SNPs that drive increased expression. (209,239,240)

(Figure 1.7A) Notably, increased copy number does not always correlate with increased expression, indicative of the critical role that post-translational modifications contribute to the stabilization of MYC in the cell.

1.6.3 Structure and regulation of MYC protein

At the protein level, MYC stability is further regulated through tightly regulated post-translational modifications. It contains several conserved regions- an N-terminal transactivation domain (TAD), that consists of a 143 amino acid domain that confers

transcriptional and cell-transforming activity along with functional modules called MYC boxes, MYC Box I (MBI) and MYC Box II (MBII). MBI houses the phosphodegron where ubiquitination and proteasomal degradation are initiated. (232,238,240,241)Generally,

MYC is considered very tightly regulated with a half-life of 20 minutes. However, in cancer cells, it has been shown that the half-life is stabilized through a combination of phosphorylation events and/or mutations that contribute to its sustained presence and activity. Specifically, a series of dephosphorylation and phosphorylation events at Serine

62 (S62) and Threonine 58 (T58) work to either stabilize or prime MYC for ubiquitination and consequent degradation by the proteasome. Phosphorylation of MYC at S62 (p-S62) through the KRAS/MEK.ERK cascade or through CDKs stabilizes MYC. Upon phosphorylation at this site, MYC Serine 63 (S63) can be isomerized from a trans to a cis conformation that enhances MYC DNA binding and transcriptional activity. Alternatively, upon phosphorylation at S62 MYC may be doubly phosphorylated at Threonine 58 (p-T58) by –kinase that initiates MYC turnover. This doubly phosphorylated state leaves MYC stable but also able to be recognized the isomerase PIN 1. Isomerization of S63 by PIN 1 from cis back to trans renders MYC accessible to PP2A. PP2A mediated dephosphorylation of T58 enables it to be ubiquitinated by the E3 ligase FBW7 followed by proteasome mediated decay. . (232,240) (Figure 1.7C). This is a tightly regulated series of events that enable MYC to respond quickly to stimuli as well as to be quickly inhibited in a controlled manner. However, in cancer cells, this is a process that is exploited to promote growth and tumorigenesis.

In solid tumors, it has been observed that MYC is stabilized through the phosphorylation of S62. In support of this, there is a greater percentage of p-S62 MYC than

of p-T58 MYC in tumors, and in patients' tumors, MYC has been found to be mutated at

T58, leading to a constitutively active form(225,242-248). Additionally, PP2A mediated dephosphorylation of MYC is a critical event for its degradation and MYC activity is further stabilized in the absence of PP2A. Lastly, as previously mentioned, MYC stability is enhanced downstream of oncogenic drives like KRAS and HER2. The combination of

MYC with other notable oncogenes further enhances MYC stability regardless of whether it is amplified or stabilized through a post-translational modification.

1.6.4 Approaches to target MYC at the transcriptional level

Given the inherent challenges of targeting a transcription factor like MYC, epigenetic targeting of its transcription has been proposed as an alternative therapeutic approach. This approach has resulted in many strategies to silence genes including inhibitors of histone deacetylases, histone methyltransferases, histone demethylases, DNA methyltransferases, and bromodomain and extra-terminal motif (BET) bromodomains.(242) All of these approaches have so far shown some indirect inhibition of MYC. BET inhibitors are perhaps the best studied in regards to inhibition of MYC activity and for their use in MYC dependent tumors. BET inhibitors primarily target the

BET family member BRD4 which initiates transcriptional elongation through the recruitment of elongation factors P-TEFb to enhancers and promotors (249,250)(Figure

1.7B). In regards to MYC activity, BET inhibition inhibits BRD4 binding at acetylated histones within the MYC promoter. This approach results in the inhibition of c-MYC but

L- and N-MYC as well. In vitro and in vivo, this approach has been promising demonstrating inhibition of cancer cell growth and anti-tumor activity(251-257). However,

in clinical trials, this approach has not resulted in prolonged anti-cancer activity, leading to disease progression and resistance when used as single agents. However, further research in vitro, in vivo, and from clinical trials has determined that these agents may sensitize cancers to some kinase inhibitors and there is a large body of evidence suggesting that employing combination strategies may be more successful than the use of bromodomain inhibitors as a single agent in MYC dependent tumors(258-261).

Another alternative to targeting the transcription of MYC that is more direct is through the targeting of DNA structures called G-quadruplexes(262-264). These structures lie upstream of a gene's transcriptional start site and when stabilized will silence a gene's expression. Site-specific molecules can be designed to G-quadruplexes associated with particular genes. There are a number that have been shown to target and stabilize the g- quadruplexes in the nuclease hypersensitive element (NHE) of the MYC promoter (Figure

1.7B). It has been shown that these molecules (GQC-05, Cz1, IZCZ-3, and DC-34) decrease MYC mRNA and consequently MYC protein. Additionally, this effect is coupled with an increase in cell death in vitro(265-269). Similarly, targeting the same concept, the small molecule stauprimide stabilizes g-quadruplexes by inhibiting the transcription factor

NME2 from being recruited to the NHE region of MYC and similarly inhibiting it.(270)

1.6.5 Inhibition of MYC protein and activity

Inhibition of Translation

In addition to advances made to inhibit MYC at the transcriptional level, a myriad of approaches have been proposed and demonstrated to target MYC protein. One approach has been to inhibit the translation of MYC altogether. The PI3K/AKT/mTOR pathway

regulates the transcriptional binding protein 4EBP1via phosphorylation of eIF4E. This event initiates translation of MYC and inhibition of the upstream pathway using inhibitors to AKT and mTOR has shown to have efficacy in MYC driven cancers. (Figure 1.7 C)

(271)

Inhibition of MYC/MAX Interaction

A variety of approaches to directly target the MYC protein itself have been proposed as well. These approaches include inhibiting MYC and inducing degradation of the MYC protein altogether. The bHLHZ domain of MYC is required for binding to MAX and downstream transcriptional activity. Thus, there have been screening and drug development research aimed at targeting this domain and interrupting MYC binding to

MAX. The potential for this approach was demonstrated initially through molecules identified through chemical library screens, 10058-F4 and 10074-G5 (Figure 1.7C). These molecules were shown to disrupt MYC/MAX binding and inhibit the transcriptional activity of MYC, but they were considered to have low potency and a number of off-target effects(272-274). More recently, two newer molecules have been identified that further demonstrate the potential of this approach and improves upon the limitations of the previously identified molecules, MYCMI-6 and KI-MS2-008 (Figure 1.7C)(275,276).

MYCMI-6 binds the bHLHZ domain of MYC but at a lower concentration (low uM) than the previously mentioned molecules. In support of this approach, in a panel of 60 cell lines, the majority (75%) of lines with high MYC mRNA or protein were sensitive to this molecule. However, while this approach may inhibit the transcriptional activity of

MYC/MAX dimerization, it does not result in a decrease of the MYC protein and more

research needs to be done to understand the potential consequences of MYC activity independent of MAX(275).

Alternatively, KI-MS2-008 inhibits the transcriptional activity of MYC by stabilizing MAX homodimers. MAX/MAX binding does not result in the output of a transcriptional program and MYC transcriptional activity is inhibited. Furthermore, in the absence of a binding partner, MYC is degraded in cells treated with KI-MS2-008. The anti- cancer effect of KI-MS2-008 has been shown both in vitro and in vivo and further demonstrates that inhibition of MYC transcriptional activity has potential as a therapeutic option for the treatment of MYC dependent or driven cancers(276).

Lastly, the inhibition of MYC activity has also been targeted through a mini-protein designed as a dominant-negative form of MYC, known as Omomyc (Figure 1.7C). This mini-protein acts by binding to MAX in the place of endogenous MYC but results in a transcriptionally inert dimer thus inhibiting MYC activity. (277-281)Moreover, in the presence of Omomyc, much like in the setting of KI-MS2-008, endogenous MYC is unstable, as it is unable to bind to Max and exert its activity.

Targeting MYC Phosphorylation

In addition to inhibition of MYC transcriptional activity through disruption of its binding with MAX, MYC protein and activity may be modulated by targeting the post- translational modification responsible for MYC stabilization. Based on the known lifecycle of MYC this could be through the modulation of phosphorylation events that contribute to MYC stability or through methods that enhance MYC ubiquitination and result in its turnover.

Phosphorylation of MYC could be regulated through two mechanisms- through the upstream kinases responsible for its phosphorylation or potential through the phosphatase activity that ultimately leads to its degradation. ERK, CDK2, and CDK9 inhibition have all been shown to inhibit MYC S62 phosphorylation (Figure 1.7C). However, it has also been shown that MYC dependent cancer cells are capable of reprogramming themselves to overcome this initial inhibition of S62 phosphorylation by kinase inhibitors leading to resistance. (220,224,225,232,240,282) Therefore, it has been proposed that promoting dephosphorylation of S62 MYC by PP2A may be another tractable approach. To date, this has been achieved through a variety of modalities that indirectly activate PP2A in cancer cells and lead it to dephosphorylate MYC. Specifically, inhibition of CIP2A and SET as described in section 1.5.1 have been shown to result in the degradation of MYC and impeded cancer growth in models of breast cancer, leukemia, and lymphoma

(244,245)(Figure 1.7C). These approaches demonstrate the potential of activating PP2A to target MYC but may be associated with off-target effects and limitations of the approaches currently in use to inhibit CIP2A and SET. But this work does lay a foundation demonstrating that activation of PP2A may be an ideal approach for targeting MYC.

1.7 Statement of Purpose

PP2A is one of the most abundant cellular proteins, a prominent phosphatase tumor suppressor that regulates the activity of numerous kinases. With a high degree of sequence conservation among yeast, drosophila, and mammals, PP2A controls many cellular functions ranging from metabolism, cell cycle, DNA replication, growth, and apoptosis. It is commonly dysregulated and deactivated in a variety of cancers and other diseases.

Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by a mediator that is downstream of the oncogenic drivers that initiate and drive cancer progression.

Direct small molecule activation of PP2A is a documented and demonstrated approach to restoring its function. The remaining challenge is to identify the contexts where this approach may be most susceptible to its activation. One such context is cancers driven or dependent on the MYC oncogene. MYC is one of the best characterized substrates of PP2A, specifically the A-B56a-C holoenzyme and previous studies have shown that indirect activation of PP2A can impede the growth of MYC driven cancers.

Thus this work strives to demonstrate that direct activation of PP2A with SMAPs is a viable approach to target MYC and inhibit tumor growth.

Important to this work is demonstrating the SMAP mediated activation of PP2A results in the inhibition of cancer cell growth in vitro and in vivo. Moreover, given the complexity with which MYC is regulated and the challenges inherent to targeting it, this works demonstrates that regardless of how its expression is enhanced- through transcriptional overexpression or stabilization at the level of the protein, SMAPs inhibit cancer cell and tumor growth and this inhibition of cancer activity is dependent on inhibition of MYC. This work uses a combination of biochemical and genetic approaches, in both in vitro and in vivo models to demonstrate inhibition of cancer activity is dependent on MYC inhibition and PP2A activation and that direct activation of PP2A is a strong therapeutic approach for the treatment of MYC driven cancers.

Figure 1.1 PP2A is a heterotrimeric complex consisting of a scaffolding subunit (A), a regulatory subunit (B) and a catalytic subunit (C). PP2A A subunit is composed of 15 tandem HEAT repeats in two isoforms, alpha and beta. PP2A C subunit also exists in two possible isoforms,alpha and beta. PP2A B subunit consists of four classes: B(B55/PR55), B’ (B56/PR61), B’’ (PR48/PR72/PR130) and B’’’ (PR93/PR110).

Figure 1.2 Structures of PP2A core enzyme and holoenzyme. (A) (PDB code 2IE3) Core enzyme consisting of Aa (in magenta) subunit and Ca (in yellow) subunit. The C subunit binds A at HEAT repeats 11–15. The active site of the C subunit consists of two manganese atoms and is positioned away from the ridge of the A subunit HEAT repeats. Binding with the catalytic subunit shifts HEAT repeats 13–15 by 20–30 _A. (B) (PDB code 3DW8) Core enzyme binding to B family subunit (in cyan), Ba/PR55a. Members of this subunit family bind the A subunit at two interfaces. The first is via a seven-bladed propeller, composed of WD40 repeats. The bottom face of the propeller binds to A subunit HEAT domains 3–7. The second is through a b-hairpin handle that interacts with A subunit HEAT repeats 1 and 2. Upon binding to the holoenzyme, the B subunit substrate binding site lies on the top face proximal to the active site of the catalytic subunit. (C) (PDB code 2IAE) Core enzyme binding to B’ family subunit (in cyan), B1/PR611. The B0 structures are similar to the A subunit, composed of eight HEAT-like repeats. These interact with HEAT repeats 2–8 of the A subunit and with the C subunit. Much like binding to B family subunits, the substrate binding site of B0 is proximal to the active site of the catalytic subunit upon holoenzyme formation. Binding to the B’ subunits forces the N-terminal repeat of the A subunit to twist 50–60 _A, rearranging the hydrophobic core of the scaffolding subunit. (D) (PDB code 4I5L) Core enzyme binding to B’’ family subunit PR72 (in cyan). B’’ subunits consist of a linear arrangement of different functional motifs that include an N-terminal hydrophobic motif and two EF hand calcium binding motifs. The N-terminal hydrophobic motif and one EF hand bind to the A subunit at HEAT repeats 1–7 and bind to the catalytic subunit via a helix on the subunit at residues 439–446 near the active site, positioning the substrate binding site near the active site. The resulting conformation of this holoenzyme is wider and taller than that which forms with B or B’ subunits.

Fig. 1.3 PP2A mutations in cancer (A) Pie chart of the frequency of PP2A mutations across 9759 tumor samples. Mutational information was analyzed from cbioportal.org, which includes 85 different sequencing studies, including the Cancer Genome Atlas (TCGA) data. Studies with targeted sequencing or expression only data were excluded from the total number. (B) Pie chart of the frequency of PP2A mutations divided by PP2A subunit families: A, B, B0, B0 0 and C. Bold black lines divide each subunit. Mutational information was analyzed from cbioportal.org, which includes 85 different sequencing studies, including TCGA data. Studies with targeted sequencing or expression only data were excluded from the total number. The results shown here are in part based upon data generated by the TCGA Research Network:http://cancergenome.nih.gov/.

Table 1.1: PP2A subunit alterations in cancer Subunit Gene Isoform Alteration Disease Reference

breast, lung, (41), (43), melanoma, ovarian, Point mutation (44), (45), endometrial, uterine, (46), (47) A PPP2R1A Aα colon

Deletion Breast (41) Decreased expression Glioma (48) (41), (51), Missense mutation breast, colon, lung (283), (284), (40) In-frame deletion breast (40)

breast, lung, ovarian, (51), (285) A Ab LOH cervical, melanoma, PPP2R1B NHL, CLL

Decreased expression AML (69)

Abberant transcription HCC, B-CLL (53),(54)

breast, prostate, Deletion (58), (57), (59) B myeloma PPP2R2A B55α Decreased expression AML (64) DNA B PPP2R2B B55b breast, colon (65), (66) hypermethylation

(63), (286), B PPP2R2C B55g Decreased expression breast, prostate (287)

B¢ PPP2R5A B56α Decreased expression melanoma (61) Decreased expression melanoma (62) B¢ PPP2R5C B56g Point mutation lung (56)

B¢ PPP2R5E B55e SNP soft tissue sarcoma (288)

(69), (70), C PPP2CA Cα Decreased expression AML, prostate (68), (67)

Fig. 1.4 Post Translational Modifications of PP2A (A) The C terminus of the catalytic C subunit undergoes methylation at L309 via LCMT, a SAM-dependent methyltransferase. PME-1 mediatesdemethylation. (B) Decreased methylation at L309 and increased phosphorylation of Y307 and T304 of the catalytic C subunit are post- translational modifications that inhibit PP2A.

Fig. 1.5 Structure of PP2A in complex with PME-1 and LCMT (A) (PDB code 3C5W) Structure of PP2A and PME-1 complex with scaffold subunit in magenta, catalytic subunit in yellow and PME-1 in red. (B) (PDB code 3P71) Structure of PP2A and LCMT complex with catalytic subunit in yellow and LCMT in green. (C) (PDB code: 4LAC) Structure of PP2A and PTPA complex with scaffold subunit in magenta, catalytic subunit in yellow and PTPA in orange.

Fig. 1.6 Approaches to activate PP2A . Several strategies to activate PP2A include, inhibiting endogenous inhibitors (SET and CIP2A), inhibiting PME-1 and using promethylating agents.

Fig. 1.7 Mechanisms of c-MYC regulation exploited in cancer and approaches to target its expression and activity. (A) Mechanisms of MYC amplification in cancer.(B)rRegulation of MYC expression highlighting super enhancer elements and g- quadruplexes, two elements upstream of the MYC gene that regulate its expression. BET bromodomain inhibitors have shown to inhibit MYC expression through the inhibition of superenhancer activity. G-quadruplex inhibitors designed specifically to a G-quadruples in the MYC promoter region have also demonstrated inhibition of MYC. (C) Schematic of MYC protein, highlighting regions important to its stability, DNA binidng and activity

and approaches to therapeutically target these regions. Also highlighted is approaches to inhibit translation of the MYC protein itself by inhibition of the PI3K/AKT/mTOR pathway.

Fig. 1.8 Schematic detailed regulation of MYC protein highlighteing the sequential steps regulating it stability, activity, and degradation,

CHAPTER 2: Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers

This research was originally published in the Journal of Biological Chemistry.

Caroline C. Farrington, Eric Yuan, Sahar Mazhar, Sudeh Izadmehr, Lauren Hurst, Brittany L. Allen-Petersen, Mahnaz Janghorban, Eric Chung, Grace Wolczanski, Matthew Galsky, Rosalie Sears, Jaya Sangodkar, Goutham Narla Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers

J. Biol. Chem. 2019 doi: 10.1074/jbc.RA119.011443jbc.RA119.011443.

2.1

ABSTRACT

The tumor suppressor PP2A is a serine threonine phosphatase whose activity is inhibited in the majority of human cancers. One of the best characterized PP2A substrates is MYC whose overexpression is commonly associated with aggressive disease. PP2A directly dephosphorylates MYC resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC driven cancers, we used a series of first-in-class Small Molecule Activators of PP2A (SMAPs) in Burkitt’s Lymphoma,

KRAS mutant non-small cell lung cancer, and triple negative breast cancer. In all tested models of MYC driven cancer, SMAP treatment results in rapid and sustained inhibition of MYC expression through proteasome mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and inhibition of tumor growth.

Importantly, we generated a series of cell lines expressing PP2A dependent phosphodegron mutants of MYC, and demonstrated that the anti tumorigenic activity of SMAPs is dependent on MYC degradation. Collectively, the research presented here demonstrates a pharmacologically tractable approach to drive MYC degradation using direct small molecule activators of PP2A for the treatment of a broad range of c-MYC driven cancers.

2.2 Introduction

The oncogene MYC is frequently amplified or over expressed in many cancer types, independent of histological subtype, and increased MYC expression is correlated with both more aggressive disease and resistance to standard of care treatments (220,289-292).

Moreover, studies have demonstrated that c-MYC (MYC) drives transformation, tumor

growth, and metastasis (218,232,292). As a result, MYC is a well validated and desirable drug target. However, despite its well validated role as a driver oncogene, MYC has gained a reputation for being “undruggable” due to its lack of a defined and structured ligand binding site, nuclear localization, and its complex regulation at the transcriptional and post- translational levels (293). In attempts to target MYC, researchers have developed alternative approaches to antagonize MYC activity including the development of Omomyc, a dominant negative form of MYC (277-279,294-296), and bromodomain inhibitors which have been well characterized to inhibit the transcription of MYC and MYC target genes through their inhibition of super enhancer elements (251,253-257). Other direct and indirect approaches have also been developed which have been very thoroughly reviewed in the literature (242,281,293). Overall, these efforts highlight the central dependency of cancers on MYC signaling and demonstrate that targeting MYC function could represent an attractive approach for the treatment of a broad range of cancers. Here, we present a therapeutic strategy that promotes the rapid degradation of MYC protein, resulting in tumor inhibition and robust inhibition of MYC transcriptional activity.

Protein Phosphatase 2A (PP2A) is a well characterized tumor suppressor, whose inactivation is critical for cellular transformation and whose activity is functionally inhibited by a diverse range of mechanisms in a wide variety of cancers (297-303). Its tumor suppressive activity is exerted through its ability to dephosphorylate a number of substrates involved in the regulation of cell growth and survival, including the MYC protein (240,241,304,305). MYC activity is dynamically regulated through a series of phosphorylation and dephosphorylation events, which allow for the rapid induction of

MYC-dependent transcription. PP2A mediated dephosphorylation of MYC at serine 62

leads to its degradation through the ubiquitin-proteasome pathway and provides essential negative regulation of this potent oncoprotein. In cancer, however, MYC is often aberrantly phosphorylated at S62 leading to its stabilization and increased activity (232,240,305). As such, we hypothesized that if the tumor suppressive activity of PP2A could be re- engaged/re-activated in cancer cells, this may prove to be a viable approach to the therapeutic targeting of MYC in cancer models.

While PP2A activation as a strategy to target MYC has been proposed and demonstrated previously, these studies relied on indirect methods of activating PP2A

(244,245,293,306-314). Here, we utilized a direct approach of targeting PP2A using Small

Molecule Activators of PP2A or SMAPs. These molecules have been well characterized for their target specificity, toxicology, and antitumorigenic properties in vivo(315-319).

Specifically, binding studies have demonstrated that these molecules bind to the A subunit scaffold of PP2A and biological assays have demonstrated that the ability to bind to PP2A is necessary to achieve their antitumorigenic effects. The target specificity of these small molecules has been extensively studied and reported on previously (316-318). Moreover, no visible toxicities have emerged in previous in vivo studies as measured by gross behavioral observations, a lack of weight loss, and no perturbations to serum chemistries or complete blood counts (CBC) in chronically treated mice (315-319). We hypothesized that given the negative regulation of MYC by PP2A, SMAP mediated activation of PP2A may represent a unique therapeutic strategy for targeting MYC signaling in cancer cells.

Recent publications have used SMAPs to demonstrate PP2A mediated changes to

MYC in specific tumor types. (271,316). However, a comprehensive attempt to profile the ability of PP2A reactivation to treat MYC driven cancers has yet to be studied. Here, we

show that direct activation of PP2A by SMAPs in vivo results in tumor growth inhibition across numerous models of MYC driven cancers and extend previous findings beyond just subcutaneous xenotransplanted models of cancer. Additionally, this current work demonstrates SMAPs inhibit MYC signaling regardless of the mechanism by which MYC drives any given cancer. We selected models based on both prevalence and mechanism by which MYC activity is increased - specifically, Burkitt’s Lymphoma (MYC genetic amplification), KRAS mutant non-small cell lung cancer (post- translational stabilization of MYC) and triple negative breast cancer (MYC overexpression). Furthermore, we utilized three distinct SMAP molecules, SMAP 1, SMAP 2, and SMAP 3 (Figure 2.1) (318) to further validate our hypothesis that PP2A activation may drive tumor growth inhibition in MYC driven cancers. These molecules are structurally similar PP2A specific activators whose difference lays primarily in their relative potency and pharmaceutic properties

(44,46) and the use of multiple independent small molecule PP2A activators rules out potential off target effects of any individual molecule, providing further evidence that the observed biology is PP2A dependent. In addition to the tumor growth inhibition noted in all models, molecular analysis of treated tumors revealed a significant decrease in total

MYC protein expression and a corresponding decrease in MYC signaling, confirming decreased MYC activity in both cellular and in vivo models. Furthermore, we demonstrate that the decrease in MYC protein levels results from changes in MYC protein stability as a result of proteasome mediated protein degradation upon PP2A mediated dephosphorylation. In support of this finding, tumors expressing mutations in the phosphodegron of MYC were no longer responsive to SMAP treatment. Collectively, our

data show direct activation of PP2A is a promising therapeutic strategy for the treatment of MYC driven cancers.

2.3 Results

2.3.1 SMAPs inhibit tumor growth in c-MYC driven Burkitt’s Lymphoma

Burkitt’s Lymphoma is a disease that has been shown to almost universally be driven by MYC as result of one of three translocations that drives high MYC expression and activity(320). As a result of this driver event, there is little genetic heterogeneity in this model making it an ideal system to study the therapeutic potential of PP2A reactivation for the treatment of MYC driven cancers. Therefore, to investigate whether direct small molecule mediated activation of PP2A inhibits tumor growth in MYC driven cancers, we treated a xenograft model of Burkitt’s Lymphoma with SMAPs and monitored tumor growth. To establish efficacy in this model, we used the Burkitt’s Lymphoma cell line

Daudi in a subcutaneous xenograft model (Figure 1A). Mice were treated with 15mpk pf

SMAP 1 twice a day (BID) per previously published and unpublished data using a range of SMAPS from 0.1-50 mpk dosed either twice a day (BID) or once a day (QD) demonstrated that doses between 5-15 mpk BID resulted in optimal tumor growth inhibition(271,316-319). Treatment with higher SMAP concentrations while well well tolerated in mice did not result in significant difference in response, thus we have used

15mpk in the majority of studies presented here. In this model, SMAPs inhibited tumor growth by approximately 65% (Figure 2.2, A and B). As has been shown previously

(317,318), SMAP 1 treatment was well tolerated with no visible toxicities or changes in body weight noted over the entire treatment course (data not shown). At the end of the

study, control and SMAP treated tumors were analyzed for phosphorylation of MYC at serine 62 as well as total MYC expression and apoptosis by immunoblot and TUNEL staining respectively. Phosphorylated MYC and total MYC protein expression were determined to be significantly decreased in SMAP treated tumors compared to control tumors (Figure 2.2, C and D) Additionally, TUNEL staining analysis revealed a significant increase in apoptosis in the SMAP treated tumors compared to control tumors (Figure 2.2E and F). Taken together, these data established the efficacy of SMAP treatment in the

Burkitt’s lymphoma Daudi model, suggestive that this is a viable approach for targeting

MYC and driving an anti-tumorigenic response as a result.

2.3.2 SMAPs decrease tumor burden and c-MYC expression in a KRAS model of

Non-Small Cell Lung Cancer

Next, we extended our studies to a genetically engineered mouse model (GEMM) model of KRAS driven non small cell lung cancer (NSCLC), KRASLA2(321). It has been established that MYC and KRAS cooperate to drive tumorigenesis and that KRAS mutant tumors can be dependent on MYC for their survival (212,322,323). MYC has been shown to be overexpressed broadly in up to 70% of NSCLC and MYC overexpression is associated with a poor prognosis (324-326). In KRAS mutant NSCLC specifically, MYC protein is further stabilized by a KRAS downstream kinase, ERK, via phosphorylation at serine 62, the same site previously described to be dephosphorylated by PP2A

(224,225,240,295,327). Through this mechanism, MYC is a critical effector of KRAS mediated activity and could represent a potential drug target for the treatment of KRAS driven cancers (295,327). We therefore hypothesized that PP2A reactivation could

represent a novel strategy for the treatment of KRAS mutant lung cancer through its negative regulation of MYC. We began by determining the expression of MYC in the tumors from the KRASLA2 (321) genetically engineered mouse model (GEMM) of lung cancer versus wildtype mice using immunohistochemistry (IHC) (Figure 2.3 A).

Importantly, there was a significant increase in MYC expression in the KRAS mutant mice compared to control mice, supporting the findings that MYC protein is stabilized by KRAS dependent signaling. We next treated the KRASLA2 GEMM of lung cancer with SMAP 2 for 28 days. At the end of the study, mice were sacrificed and the lungs were removed for downstream molecular and histological analysis. Consistent with previous results, overall tumor volume was significantly decreased in SMAP 2 treated mice compared to control, consistent with a marked reduction in lung cancer nodules as measured by histological quantitation (Figure 2.3, B-D). The observed reduction in tumor growth was associated with an increase in apoptotic signaling in the tumor nodules of SMAP 2 treated mice compared to control as measured by TUNEL staining (Figure 2.3B and Figure 2.4A) and a decrease in MYC expression (Figure 2.3 B). This data provides further support to our hypothesis that PP2A activators may have robust anti-tumor activity through their ability to regulate MYC expression and signaling.

In addition to the KRASLA2 model of non-small cell lung cancer, we also assessed response to SMAP treatment in a subcutaneous xenograft model of KRAS driven lung cancer using the H441 cell line which harbors a KRAS G12V mutation. We have previously shown that SMAPs have activity in H441 in vitro.(318) To determine if SMAP treatment inhibited H441 tumor growth in vivo, we performed a subcutaneous xenograft and treated with SMAP 1. We found SMAP 1 significantly inhibited tumor growth by

approximately 76% (Figure 2.3, E and F). Additionally, tumors treated with SMAP 1 showed a significant increase in TUNEL positivity indicative of apoptosis when compared to control tumors (Figure 2.3, G and H). Finally, we analyzed MYC expression by IHC and, as hypothesized, greater MYC expression was seen in tumors from the control group, supportive of the coupled relationship between KRAS and MYC activation (Figure 2.3 I).

Consistent with our results with the KRASLA2 GEMM model, tumors treated with SMAP

1 had lower phospho s62 and total MYC expression as determined by both IHC and western blot (Figure 2.3 , I and J, Supplemental Figure 3.4, B, C and D). Combined, these findings suggest that the therapeutic efficacy of small molecule mediated reactivation of PP2A may be a result of coordinate down regulation of both MYC and KRAS signaling.

2.3.3 SMAPs inhibit tumor growth in c-MYC expressing xenograft models of TNBC

To extend the translational impact of our findings, we investigated the potential of

SMAPs to inhibit tumor growth in xenograft models of triple negative breast cancer

(TNBC). TNBC is a breast cancer subtype that is notoriously aggressive and less responsive to current standards of care treatment. MYC has been well described to be commonly overexpressed and functionally active in claudin low and basal like TNBC

(325). Moreover, high MYC expression in these subtypes is associated with poor response to current standard of care treatments (328). It has been shown that the high expression of

MYC contributes to a state of MYC oncogene addiction in these cancers and as such has been well described as a potential drug target for the treatment of TNBC (245,325,329-

331). In support of this, it has previously been shown that direct or indirect inhibition of

MYC inhibits disease progression in both in vitro and in vivo models of the disease

(245,255,330,332).

First, we performed in vitro studies to test SMAP response in a series of TNBC cell lines, MDA-MB-231, MDA-MB-453, BT549, and HCC1937. All lines were sensitive to

SMAP treatment as demonstrated by dose dependent decreases in cell viability and reduced colony formation in a mammosphere assay (data not shown). Moreover, in the mammosphere assay, HCC1143 appears to be more sensitive to SMAPs than to the standard of care for TNBC, Paclitaxel, as well as a MEK inhibitor, AZD6244, which has previously been proposed as a targeted therapy approach for TNBC (333,334).

To extend these findings to disease relevant in vivo models, we tested SMAP efficacy in a series of TNBC xenograft models. We selected three TNBC cell lines, previously characterized for their high MYC expression and MYC dependency for efficacy testing with our small molecule PP2A activator series. The MDA-MB-231 cell line is a claudin low TNBC cell line that harbors a KRAS mutation that is proposed to contribute to MYC stability much like the lung cancer models described above (325). The SUM149PT line is a basal like TNBC cell line which demonstrates MYC dependency (329). Finally, we used the MDA-MB-453, a TNBC cell line that expresses the androgen receptor (AR) and high levels of the MYC protein. Importantly, much like has been shown in prostate cancer, there is a strong relationship in breast cancer between MYC and AR pathway activation and increased tumorigenesis and drug resistance (335,336). Treatment with

SMAP 1 induced tumor growth inhibition of 46% in the MDA-MB-231 model (Figure 2.5

A and B), 68% in the MDA-MB-453 model (Figure 2.5 E and F) and 69.7% in the

SUM149PT model (Figure 2.5 G and H). Again, no significant weight loss or visible

toxicities were identified in the treated mice consistent with previous in vivo studies performed (data not shown). In the MDA-MB-231 model, the anti-tumor effects of SMAP

1 was comparable to that of Paclitaxel (Figure 2.5 A and B) and SMAP treated tumors demonstrated decreased phosphorylation of MYC at serine 62 as well as a decrease in total

MYC protein expression (Figure 2.5 C and D). Importantly, in the MDA-MB-453 model, the majority of tumors regressed upon treatment with SMAPs (Figure 2.5 D). As a result of their small size or lack of remaining tumor, MYC expression could not be assessed in

SMAP treated tumors from the MDA-MB-453 or SUM149PT models. Collectively, our findings demonstrate that SMAP treatment inhibits tumor growth in multiple models of

MYC-expressing TNBC.

2.3.4 SMAP treatment results in proteasome mediated MYC degradation

To determine the mechanism by which PP2A activation alters MYC expression, we treated Daudi cells with SMAPs and analyzed MYC stability and function. Upon SMAP treatment, both phosphorylated MYC (s62) and total MYC protein expression rapidly decreased, with a greater than 50% loss seen within 2 hours and minimal protein remaining by 4 hours (Figure 2.6, A,B,C). To determine if these changes were occurring post- transcriptionally, we analyzed the mRNA levels of MYC after treatment with SMAPs and found no significant change (Figure 2.7A), indicating the decrease in MYC expression resulted from post-transcriptional mechanisms of regulation. In order to determine the generalizability of these findings, we assessed MYC protein expression upon PP2A activation in two of the TNBC cell lines used in vivo, MDA-MB-453 and MDA-MB-231.

Consistent with the results in the Daudi cell line, MYC protein expression decreased within

3 hours (Figure 2.7 B,C). Additionally, we assessed MYC half life in the MDA-MB_231 cell line in the presence and absence of SMAPs using cycloheximide and found that

SMAPs shortened MYC half life by nearly 50% from 35 minutes to 18 minutes(Figure 2.6

D, E, F).

Multiple publications have demonstrated that MYC stability is regulated by ubiquitination through several E3 ligases, including FBXW7. Recognition of MYC by

FBXW7 is directed by PP2A mediated S62 dephosphorylation, triggering MYC degradation by the proteasome (232,240,305,337). To determine if the SMAP mediated decrease in MYC protein expression was proteasome mediated, we treated cells with

SMAP 1 in the presence of the proteasome inhibitor MG132. Daudi cells were pretreated for two hours with DMSO or MG132, and then treated with 20 µM of SMAP or DMSO for 2 hours. Consistent with previous findings, total MYC expression decreased in the

SMAP treated cells (Figure 2.6 G,H). However, in cells that were pretreated with MG132, these changes were significantly abrogated, indicating the decrease in MYC expression in response to SMAP 1 resulted from proteasome mediated degradation of the protein (Figure

2.6, G, H).

In summary, we show that the decrease in MYC protein expression upon PP2A activation occurs post translationally and that this decrease could be rescued through inhibition of the proteasome consistent with the established mechanism of MYC degradation upon PP2A activation. This data, combined with the established mechanism of MYC degradation, demonstrates that small molecule activation of PP2A results in the degradation of MYC by the proteasome.

2.3.5 SMAPs inhibit the transcription of c-MYC target genes

Based upon the observed changes in MYC protein expression, we next sought to confirm the loss of protein by deteriming if its activity was changed. A panel of well described transcriptional targets of MYC was selected from the literature (Table 1.1)

(338,339) and changes in their expression was assessed after SMAP treatment in the Daudi and MDA-MB-231 cell lines. In both cell lines, 21 of 23 of the MYC gene targets, that are reported to be transcriptionally upregulated by MYC, were downregulated with SMAP treatment (Daudi: Figure 2.6 F, Table 2.2, MDA-MB-231: Supplemental Figure 2.7D,

Table 2.3). Conversely, two genes shown to be transcriptionally inhibited by MYC were also assessed after SMAP treatment and of these two targets, one (CEBPA) was signficiantly upregulated in both cell lines. Overall, these changes in mRNA expression of

MYC target genes demonstrate a change in MYC transcriptional output upon SMAP treatment.

Mutation of the c-MYC phosphodegron abrogates SMAP driven tumor growth inhibition

To determine if SMAP induced dephosphorylation and degradation of the c-MYC protein is responsible for its reported anti-cancer activity, we expressed a phospho-mimetic version of MYC (S62D); if SMAP mediated dephosphorylation of MYC at this defined site drives the observed anti-cancer activity, then these effects will be attenuated in the presence of the phosphodegron mutant MYC S62D. To test this functional dependency, we expressed EGFP as a control, wt-MYC, MYC S62D, and a Threonine 58 to Alanine (T58A)

MYC mutant (Figure 2.8 A and B) which allows for PP2A dependent dephosporylation at

S62, but inhibits downstream degradation of MYC to occur by preventing c-MYC from being identified by its E3 ligase.

As previously demonstrated in the parental Daudi line, SMAP treatment inhibited tumor growth in the Daudi EGFP cell line (47%), induced tumor cell apoptosis as quantitated by TUNEL staining, and drove decreased MYC expression as observed by western blot of tumor lysates (Figure 2.9, A-E). This same trend was seen in the line overexpressing WT MYC, although the effect here was modestly abrogated (31% tumor growth inhibition) (Figure 2.10, A-E). The overexpression of MYC generally promotes more aggressive tumor growth as observed by the differences in growth between this model and parental Daudi or Daudi EGFP and as a result this may have affected the magnitude of

SMAP response. Interestingly, in both the Daudi MYC S62D xenograft and the Daudi

MYC T58A xenograft, there was no tumor growth inhibition by SMAPs (Figure 2.9F,

Supplemental Figure 2.10F). Additionally, in both of these models, SMAP treatment did not result in any significant induction of apoptosis as assessed by TUNEL staining, and

MYC protein expression was unchanged in these MYC mutant xenografts (Figure 2.9, G-

J, Supplemental Figure 2.10, G-J).

When assessing MYC expression across tumor lysates from these studies, there are two bands both migrating around the molecular weight (MW) reported for MYC that were not previously seen in vitro. To determine if a single or both bands should be used to quantitate MYC expression, a blot was run with representative tumor lysates from each study. Overall it seems that both bands were overexpressed in the models with MYC overexpression (Figure 2.11). Thus, in order to quantify MYC expression in each of the in vivo studies presented, both bands were analyzed. Additionaly, this blot demonstrates that

overexpression of MYC, MYC S62D and MYC T58A appears to have been sustained through the course of the study.

In summary, these studies demonstrate that PP2A mediated dephosphorylation of

MYC is necessary for SMAP driven tumor growth inhibition in these models and solidify the proposed mechanism of PP2A in regulating MYC S62 phosphorylation and degradation.

2.4 Discussion

This work builds on a robust body of research demonstrating that MYC is a potent oncogene and that targeting it could be an ideal therapeutic strategy in a diverse range of cancers, many of which progress rapidly on standard of care treatments and in general are associated with quite poor prognosis. In the original research presented here, we have demonstrated that the degradation of the MYC protein using a first-in-class series of direct small molecule PP2A activators has significant single agent pre-clinical activity.

Moreover, this approach has the following advantages: 1) these small molecules are orally bioavailable and well tolerated across a number of preclinical models, 2) PP2A reactivation targets MYC at the protein level resulting in its rapid degradation, essentially acting as a

MYC degrader, and 3) the specificity of these small molecules to PP2A has been extensively validated. In aggregate, this is a first-in-class and pharmaceutically tractable approach targets MYC degradation leading to a significant reduction in overall cell viability and a reduction in tumor volume in vivo.

One advantage to targeting MYC via PP2A reactivation is that it may overcome the diversity of mechanisms by which MYC is overexpressed, activated, or stabilized in the

cell. This is most likely because the negative regulation of MYC by PP2A is one of the more downstream events in MYC’s “lifecycle” and thus is not restricted to controlling only one component or step in the regulation of MYC stability. This is highlighted in the current work, as multiple models of MYC dependency were tested including: 1) genetic amplification (Burkitt’s lymphoma), post-translational stabilization (KRAS mutant cancers), and overexpression (TNBC).

Increased MYC expression is well described to alter and regulate a number of cellular processes that cancer cells become addicted to including, but not limited to: cellular metabolism, cellular proliferation, cell surivival and differentiation (293). Future studies directed at understanding the processes that cells most depend upon in the context of increased MYC expression and PP2A dysregulation could help further define cancer subtypes most susceptible to this treatment strategy as well as shed insight on potential resistance mechanisms to MYC targeting strategies.

Additionally, while MYC is one of the better characterized substrates of PP2A,

PP2A has diverse cellular substrates. As demonstrated by our studies in KRAS mutant lung cancer models, we may be able to leverage our knowledge of defined PP2A substrates to identify disease contexts that are particularly susceptible to PP2A activation through coordinate effects of both MYC signaling and other oncogenic pathways including PI3K and MAPK. Additionally, in triple negative breast cancer, a MYC gene signature associated with activation has been proposed as a potential biomarker (340). Based on the research presented here, this subset of this particularly lethal disease may be especially treatment sensitive to PP2A reactivation strategies.

Lastly, while the models presented here represent a variety of well characterized examples of MYC driven cancers, they represent only a subset of potential MYC targetable cancers. Specifically, this PP2A reactivation approach has the potential to be translated to multiple models of MYC driven cancer such as pancreatic cancer, which is similar to

NSCLC in the common co-expression of KRAS and MYC, as well as prostate cancer where

MYC has been implicated in the deregulation of the Androgen Receptor (AR) driven transcriptional programs and is associated with poor response to AR directed therapies.

Indeed, it was recently published that SMAPs syngerize with mTOR inhibition in pancreatic cancer to reduce tumor growth through decreased MYC expression (271).

Moreover, SMAPs were previously shown by our group to impact prostate cancer growth via destabilization of the androgen receptor resulting in marked changes in its downstream transcriptional activity (317). Thus, this work could lay the foundation for the use of PP2A reactivation strategies in combination with AR directed treatments to drive more durable treatment responses or as a single agent in enzalutamide resistant metastatic prostate cancer.

Overall, the work presented here expands the spectrum of cancer models in which

PP2A activation is efficacious and suggests that therapeutic PP2A activation by SMAPs may be a useful strategy for the treatment of MYC driven cancers.

2.5 Experimental Procedures

Cell lines and Reagents Burkitt’s Lymphoma cell line Daudi, lung cancer cell line,

H441, and breast cancer cell lines, MDA-MB-231, MDA-MB-453, SUM149PT, BT-549 and HCC1143 were purchased from the ATCC. H441 and Daudi cell lines were maintained

in RPMI 1640 medium (Corning Mediatech, Inc., Manassas, VA). MDA-MB-231, MDA-

MB-453, SUM149PT, BT-549 and HCC1143 were maintained in DMEM (Corning

Mediatech, Inc., Manassas, VA). All media was supplemented with 10% FBS(VWR

International, Avantor Performance Materials, Center Valley, PA) and 50 units/mL of penicillin-streptomycin solution (GE Healthcare, Little Chalfont, UK). Cells were maintained at 37C with 5% CO2 Mycoplasma testing was performed routinely with Lonza

MycoAlert Myco- plasma Detection Kit as per the manufacturer’s protocol (catalog no.

NC9922140, Thermo Fisher Scientific). SMAP compounds were diluted with DMSO to a stock concentration of 80 µM and stored at room temperature. Dilutions to the treatment concentrations were made in appropriate RPMI or DMEM accordingly. MG-132

(Calbiochem, San Diego) was dissolved and aliquoted in DMSO at a concentration of

50mM, stored at -80 and serially diluted to 10uM in media. Cycloheximide solution (100 mg/mL) was purchased from Millipore Sigma (C4859) and stored per manufacturer’s instructions.

MTT Assay. The cells were treated with the SMAPs (dissolved in DMSO) and screened for cell viability through the MTT assay using a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide kit (Sigma-Aldrich).

Mammosphere assay. Breast cancer cells were plated on ultra-low attachment plates

(Fisher) at 25,000 cells per well in 2 ml of mammosphere media (B27, bFGF (20ng/ml),

EGF (20ng/ml), Gentamycin (100ug/ml), and amino acids in DMEM/F12). Media was

replenished every 3 days. Starting at day 4 cultures were treated for 24 hours with the indicated doses of SMAP and then imaged and quantified.

Constructs and generation of recombinant cell lines. MSCV-N GFP was a gift from Karl

Munger (Addgene plasmid # 37855 ; http://n2t.net/addgene:37855 ;

RRID:Addgene_37855). MSCV h c-MYC IRES GFP was a gift from John Cleveland

(Addgene plasmid # 18119 ; http://n2t.net/addgene:18119 ; RRID:Addgene_18119). MIG-

MYC_S62D and MIG-MYC_T58A were generated from MSCV h c-MYC IRES GFP using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s protocol using the primers 5’-

ACCCCGCCCCTGGACCCTAGCCGCCG-3’and

5’CGGCGGCTAGGGTCCAGGGGCGGGGT-3’ (MIG-MYC_S62D) or 5-

AGCTGCTGCCCGCCCCGCCCCTG-3’ and 5’-

CAGGGGCGGGGCGGGCAGCAGCT-3’ (MIG-MYC_T58A). For retroviral transduction, Gryphon packaging cells (Allele Biotechnology) were transfected using X- tremeGENE HP (Roche) using a 2:1 ratio of transfection reagent to plasmid DNA.

Transfection media was replaced after 12 hours. After 30 hours, media containing viral particles was collected and directly used to transduce H358 cells. The transduced cells were sorted for GFP positivity using a BD FACSAria and MYC overexpression was confirmed by Western Blot. Daudi cells were transduced with viral supernatant in RetroNectin coated

6-well plates (Takara Bio USA, Inc.) according to the manufacturer’s protocol

(RetroNectin-Bound Virus infection method). Transduced cells were sorted for GFP as described for H358 cells.

Mouse models and treatment studies: KrasLA2 mice were purchased from the National

Cancer Institute Mouse Repository. For xenograft studies, 5 x 10^6 Daudi cells were injected subcutaneously into the right flank of 6-8 week old female Nod-Scid Mice in a 1:1 suspension of RPMI: Matrigel. 2.5*10^6 MDA-MB-231or 1*10^6 SUM149PT cells were injected orthotopically into the right mammary fat pad of female NSG mice in a 1:1 suspension of DMEM and Matrigel. 5*10^6 MDA-MB-453 cells were injected subcutaneously into male NSG mice in a 1:1 suspension of DMEM and Matrigel. 5x10^6 of H441 cells were injected subcutanesouly in the 6-8 week old male nude mice (Strain

490, National Cancer Institute) in a 1:1 suspension of RPMI: Matrigel. Tumors were measured every other day by caliper and body weight measured every four days. Mice were treated with SMAPs or Paclitaxel when average tumor volume reached about 100mm3.

Daudi, MDA-MB-231 and H441 xenografts were treated until mice had a body conditioning score of 1, tumor volume exceeded 1200mm3 or study reached 30 days respectively. SUM149PT tumors were treated for 20 days and MDA-MB-453 tumors were treated for 33 days. At termination of study, mice received a final treatment 2 hours before sacrifice. Tumor tissue was collected and both formalin-fixed, for IHC, and snap frozen in liquid nitrogen for immunoblotting and mRNA analysis.

In vivo administration of SMAPs. SMAPs were delivered by oral gavage, twice a day (BID) at 5 or 15 mg/kg in a solution of 10% N,N-Dimethylacetamid (DMA) and 10% Solutol®

HS15 (Kolliphor® HS 15) in sterile water.

In vivo administration of Paclitaxel. 20mg/kg Paclitaxel was delivered via IP injection

1X/week Paclitaxel was dissolved in DMSO followed by addition of a pre warmed 1:1 solution of sterile water and Propylene Glycol such that the final DMSO concentration was

20%.

Antibodies and Immunoblot Analyses. Cells were washed 2x in PBS upon collection and then lysed in RIPA buffer (ThermoFisher Scientific, Waltham, MA) containing phosphatase and protease inhibitors (Roche, Basel, Switzerland). Proteins lysates were separated by SDS-PAGE 12% polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were probed with anti-phospho c-MYC s62 (Abcam), anti-phospho c-MYC t58 (abcam), total c-MYC (cell signaling), gapdh (santa cruz) and vinculin (santa cruz) Primary antibodies were probed with either goat anti-mouse (Abcam, Cambridge, United Kingdom) or donkey anti-rabbit (GE Healthcare, Little Chalfont, United Kingdom) conjugated to horseradish peroxidase and imaged and quantified usng using the Bio-Rad ChemiDoc XRS chemiluminescence imager and software. All values were normalized to vinculin, gapdh, or total protein expression (using the Bio-Rad Mini-Protean TGX Stain Free pre-cast gels) and expressed as fold change relative to control.

Quantitative real-time quantitative PCR. Total RNA was extracted using the High Pure

RNA Isolation Kit (Roche, Basel, Switzerland). cDNA synthesis was carried out using the iScript cDNA Synthesis Kit (Bio-Rad) as per the manufacturer's instructions. Sequences can be found in Supplementary Table 1. Real-time PCR was performed with SYBR green

PCR Master Mix (Applied Biosystems) on the Applied Biosystems 7900HT Fast Real-

Time PCR System.

TUNEL Staining. Tissue was fixed in 10% buffered formalin phosphate (Thermo Fisher

Scientific, catalog SF100-4), transferred to 70% ethanol, and blocked in paraffin. Serial tissue sections (5-µm thick) were cut from the paraffin-embedded blocks and placed on charged glass slides. The ApopTag Fluorescein in Situ Apoptosis Detection Kit (Millipore) was used according to the manufacturer’s protocol to perform the TUNEL assay. Before the addition of terminal deoxynucleotidyl transferase enzyme, sections were deparaffinized with xylene and rehydrated through graded alcohol washes. VECTASHIELD Mounting

Medium with Propidium Iodide (Vector Labs) was used for counterstaining in the

KRASLA2 and H441 xenograft models. All other studies were counterstained for Dapi and mounted with ProLong™ Diamond Antifade Mountant (Molecular Probes # P36961)

Fluorescent images were captured using the Zeiss Axioplan 2 IE microscope.

Quantification was completed using the cell counter function of ImageJ

(http://imagej.nih.gov/ij/). Imaging was performed at the Microscopy Cores at the Icahn

School of Medicine at Mount Sinai and University of Michigan School of Medicine.

Immunohistochemistry. Tissue was fixed in 10% buffered formalin phosphate (Thermo

Fisher Scientific, catalog SF100-4), transferred to 70% ethanol, and blocked in paraffin.

Serial tissue sections (5-µm thick) were cut from the paraffin-embedded blocks and placed on charged glass slides. Tumor sections were stained with H&E and c-MYC (Abcam, ab32072), Briefly, sections were deparaffinized with xylene and rehydrated through graded

alcohol washes followed by antigen retrieval in a pressure cooker (Dako) in citrate buffer

(10 µM, pH 6.0, Vector Labs). Slides were then incubated in hydrogen peroxide/methanol, followed by incubation in normal goat serum in PBS. Antibody was applied overnight at

4°C. DAB substrate was applied followed by counterstaining in hematoxylin. Images were captured with Olympus MVX10 (H&E) or Zeiss Axioplan 2IE (IHC) microscope. Imaging was performed at the Microscopy Core at the Icahn School of Medicine at Mount Sinai.

Statistics. Analysis was performed using GraphPad Prism 7. Statistical significance was assumed for a 2-tailed P value of less than 0.05 using Student’s t test or 2-way ANOVA with Tukey’s post hoc test (presented as means; error bars indicate SEM or SD as noted in figures).

Study Approval. Animal studies were performed under protocols approved by the

Institutional Animal Care and Use Committee of the Center for Comparative Medicine and

Surgery at the Icahn School of Medicine at Mount Sinai (protocol#: IACUC-2013-

1426/Novel Small Molecules to Treat Cancer), Case Western Reserve Unviersity (protocol

#’s 2013-0130 and 2013-0132), and Oregon Health and Sciences University (protocol #

IP00001014)

2.6 Acknowledgements: The authors wish to acknowledge the contributions of Shirish

Shenolikar for insight and critical evaluation of the work. We also thank Caitlin O’Connor for assistance with the manuscript. The authors wish to acknowledge the support of the

Young Scientist Foundation. GN and MDG would like to acknowledge NIH/National

Cancer Institute (R01CA181654). This research was supported by the Athymic Animal

and Preclinical Therapeutics, as well as the Cytometry and Imaging Microscopy Shared

Resources of the Case Comprehensive Cancer Center (P30CA043703), and the Icahn

School of Medicine at Mount Sinai Microscopy Core (P30CA196521). SI is supported by the Loan Repayment Program (NIH/National Center for Advancing Translational

Sciences).

Figure 2.1. Structure of SMAP 1 and 2

Figure 2.2. SMAPs inhibit tumor growth and decrease c-MYC expression in a model of Burkitt’s Lymphoma. (A) Tumor growth in a xenograft model of Burkitts Lymphoma with the Daudi cell line treated with DMA (control) or 15 mpk SMAP 1 by oral gavage BID. (B) Change in indivudal tumor volume at end of study (C)Western blot for c-MYC expression from tumor lysates (D) quantificaiton of p-MYC (s62) and MYC expression normalized to total protein, stainfree images used for normalization are included in supplemental figure 3A) (E) Representative images of tunel staining in tumors treated with DMA or SMAPs. (F) Quantifiacation of TUNEL Respective quantifications are represented as mean ± SEM (A) or mean +/- SD (D, E) **P < 0.01; ****P < 0.0001.

Figure 2.3 SMAPs inhibit tumor growth and decrease c-MYC expression in KRAS driven NSCLC. (A) Immunohistochemistry for c-MYC expression in lung tissue of Wildtype and KRAS LA2 GEMM model of NSCLC (B) H&E of Lungs, Tunel Staining, and immunohistochemistry for c-MYC in lungs from KRASLA2 mice treated with vehicle control (DMA) or SMAP-2 (C) Percentage of tumor volume in lungs of KRASLA2 mice treated with DMA or SMAP 2 (D) number of tumor nodules in lungs. (E)Tumor growth in a H441 xenograft model of NSCLC treated with DMA or SMAP 1

(F) Change in indivudal tumor volume at end of study (G) Representative Images of tunel in tumors from H441 xenograft. (H) Quanatification of Tunel. (I)Representative images of c-MYC expression in H441 tumors by IHC (J) quantification of c-MYC expression from tumor lysates assessed by western blot in supplemental figure 3A. Respective quantifications are represented as mean ± SD. * P< 0.05 **P < 0.01; ****P < 0.0001.

Figure 2.4 SMAP treatment increases TUNEL staining and decrease c-MYC protein expression in NSCLC mouse models upon treatment with SMAPs (A) Quantificaiton of tunel staining from the KRAS LA2 mouse model of NSCLC in DMA versus SMAP 2 treatment (B)Tumor lysates from H441 xenograft treated with DMA and (C) SMAP 1 vs DMA treatment assessed by western blot for phospho c-MYC and total c-MYC protein expression. (D) Quantification of phospho-MYC expression Tumors normalized in quantification to DMA lysate 10 (quantification of total MYC shown in figure 2J). Respective quantifications are represented as mean ± SD. * P< 0.05 **P < 0.01; ***P < 0.001.

Figure 2.5 SMAPs inhibit tumor growth in models of triple negative breast cancer. (A)Change in mean tumor volume over time in xenograft models of triple negative breast cancer cell lines MDA-MB-231,(B) Western blot for phospho MYC (serine 62) and total MYC in control and treated tumors (C) quantification of phospho and total MYC expression across tumors in B. Expression levels all normalized to DMA tumor # 3 (E) MDA-MB-453 and (G) SUM149PT treated with DMA and SMAP 1 by oral gavage BID

and in MDA-MB-231 Paclitaxel 1x/week IP. (B,F,F) Individual changes in tumor volume at end of study quantified in (B)MDA-MB-231 (D)MDA-MB-453 (F) SUM149PT. Respective quantifications are represented as mean ± SD. * P< 0.05 **P < 0.01; ****P < 0.0001.

Figure 2.6 SMAPs decrease c-MYC expression through a proteasome mediated mechanism and induce changes to c-MYC target genes. (A) Western blot for phospho (s62) and total c-MYC in Daudi cell line after exposure to 20uM SMAP 1 over 1,2,4 hours (B) quantification of phospho and (C) total c-MYC protein in n=3 experiments normalized to Vinculin (D) Western blot of MDA-MB-231 cells treated with cyclohexamide (CHX) over time and calculation of MYC half life (E) Western blot for c- MYC expression in cells co-treated with SMAP 1 and cyclohexamide over time and calculation of MYC half life. (F) quantification of MYC in (D,E) over time used for calculation of c-MYC half-life in each condition and confidence interval (G) Western blot for c-MYC expression upon treatment with MG132, SMAP 1, or combination (H) quantification of (G) for an n=3 (I) Changes to mRNA expression of c-MYC target genes in Daudi cell line upon treatment with 20uM SMAP 1 for 6 hours, representative of n=4. c-MYC target genes are separated into two groups: up-regulated c-MYC target genes and

down-regulated c-MYCtarget genes. mean fold change, standard deviation, and p-value provided for each target in Table S2. Respective quantifications are represented as mean ± SD. * P< 0.05 **P < 0.01; ***P<.001 ****P < 0.0001.

Figure 2.7 . SMAPs do not induce changes to c-MYC mRNA in Burkitts or breast cancer cell lines and inhibit c-MYC protein in breast cancer cell lines (A) mRNA expression of c-MYC in Daudi cell line upon treatment with 20uM SMAP 1 at 6 hours (B) Western blot and n=3 quantification for MYC expression in MDA-MB-453 at 3 hours with SMAP 1 treatment and (C) Western blot and n=3 quantification for MYC expression in MDA-MB-231 at 3 hours with SMAP 1 treatmen. (D) Changes to mRNA expression of c-MYC target genes in MDA-MB-231 upon treatment with SMAP 1 for 6 hours

Table 2.1: c-MYC target genes used to assess c-MYC transcriptional activity and corresponding primer sequences for qRT-PCR.

Table 2.2. Changes to c-MYC target genes in the Daudi cell line. Mean (n=4) Log2(fold change) of c-MYC target genes after 6 hours of SMAP 1 exposure normalized to DMSO treated cells.

Table 2.3 Changes to c-MYC target genes in the MDA-MB-231 cell line. Mean (n=3) Log2(fold change) of c-MYC target genes after 6 hours of SMAP 1 exposure normalized to DMSO treated cells.

Figure 2.8 Confirmation of MYC overexpression in Daudi cell line (A) (A) mRNA expression for MYC in EGFP, WT-MYC, MYC S62D and T58A overexpressing cell lines and (B) corresponding expression of c-MYC protein

Figure 2.9 SMAP inhibition of tumor growth and changes to c-MYC expression is abrogated by mutation to c-MYC phosphodegron. (A) Tumor growth in a xenograft model of Daudi cell line expressing EGFP treated with DMA or SMAP 1 BID (B) Representative images of tunel staining in DMA or SMAP 1 treated Daudi-EGFP tumors (C) quantification of TUNEL in Daudi-EGFP tumors (D) Western blot of untreated and treated tumor lysates for c-MYC in the Daudi EGFP xenograft (E) quantification of c- MYC protein in DMA and SMAP treated, Daudi-eGFP tumors tumors in D, normalized to GAPDH (F)Tumor growth in a xenograft model of Daudi cell line overexpressing c- MYC with a S62D mutation treated with DMA or SMAP 1 BID (G) Representative images of tunel staining in DMA or SMAP 1 treated tumors from the s62D xenograft (H) quantification of Tunel in the S62D tumors (I) western blot of protein from tumor lysates

for c-MYC from the s62D xenograft (J) quantification of c-MYC protein in DMA and SMAP treated tumors from the s62D xenograft in I normalized to GAPDH Respective quantifications are represented as mean ± SD. * P< 0.05 **P < 0.01; ***P<.001 *

Figure 2.10 SMAP inhibition of tumor growth and changes to c-MYC expression is abrogated by mutation to c-MYC phosphodegron (A)Tumor growth in a xenograft model of Daudi cell line expressing WT c-MYC treated with DMA or SMAP 1 BID (B) Representative images of tunel staining of WT c-MYC tumors tumors treated with DMA or SMAP 1 (C) quantification of TUNEL in WT c-MYC tumors. (D)Western blotting for c-MYC in DMA and SMAP 1 treated WT c-MYC tumors (E) quantification of c-MYC protein in DMA and SMAP treated WT c-MYC tumors normalized to GAPDH (F)Tumor growth in a xenograft model of Daudi cell line expressing c-MYC with a T58A mutation

treated with DMA or SMAP 1 BID (G)Representative images of tunel staining of MYC T58A tumors treated with DMA or SMAP 1 (H) quantificaiton of TUNEL in MYC T58 tumors. (I) Western blotting for c-MYC in DMA and SMAP 1 treated MYC T58A tumors (J) quantification of c-MYC protein in DMA and SMAP treated MYC T58A tumors normalized to GAPDH

Figure 2.11 Confirmation of c-MYC band and overexpression in Daudi tumor lysates. (A) Lysates from two different tumors in each Daudi xenograft presetned in figure 5A-J and supplemental figure 8A-J, Daudi GFP, Daudi c-MYC, Daudi c-MYC s62d, Daudi c-MYC t58A for c-MYC. (B) quantification of MYC expression in A.

Chapter 3: Discussion and Future Directions

3.1 Summary

The studies described within have demonstrated an effective approach to targeting c-MYC through the direct activation of PP2A. In multiple models of c-MYC driven cancers, SMAPs are effective as a single agent therapy. Regardless of the mechanism of amplification or overexpression, this approach demonstrates that targeting MYC at the level of the protein and inducing its degradation overcomes its expression and activity. A strength of this research is the diversity of models used, GEMM and xenograft from blood and solid tumors. It further supports the hypothesis that MYC inhibition may inhibit tumor growth regardless of the context or other co-expressed oncogenes because of the dependency cells develop for it. Looking forward, the range of models used also present a unique framework for studying this relationship further, seeking to identify unique or common dependencies in cancers that overexpress MYC concurrent with PP2A inhibition.

It also lays the foundation for studying this approach in the context of cancers driven by similar oncogenes, N-MYC and L-MYC. These studies are discussed further in this section.

3.2 SMAPs for the management of N-MYC and L-MYC driven cancer

c-MYC is one of three in the MYC family of transcription factors, the other two being N-MYC and L-MYC. The Like MYC, MYCN and MYCL are important to development as well as being proto-oncogenes. They all have similar functions and have been described to drive cancers in certain settings(231,341). Unlike MYC though, their expression tends to be much more restricted and tissue specific. Whereas MYC expression has been well characterized across a diverse set of cancers, N-Myc expression is primarily expressed in neuro-endocrine tissue and L-Myc was first described in lung tissue and appears to be only expressed in lung tissue. However, while they may be associated with

fewer types of cancers, the diseases with which they are associated with are notorious for being difficult to treat and for being rather aggressive.(341)

N-MYC

MYCN is primarily expressed during embryogenesis. Its expression is highest in the developing brain but has been observed in other tissue including pre-B cells, kidney, forebrain, hindbrain, and intestine. It is downregulated after embryogenesis and its expression is rare in adult tissue(342). The cancer that N-Myc is most associated with is neuroblastoma- the setting where it was originally discovered. Neuroblastoma is a disease that primarily affects children. And in many instances, it responds very well to the current standard of care or regresses on its own. However, about 20% of neuroblastomas have been shown to have N-Myc amplification. This subset of neuroblastomas is further correlated with more aggressive disease as well as poor response to current treatment options(342,343). N-Myc is also amplified or overexpressed in other tumors of neuronal origin- in about 5% of medulloblastoma, a small subset of glioblastoma multiforme, the majority of retinoblastomas, and rhabdomyosarcoma(342,344-347). Similarly to neuroblastoma, N-Myc expression in these diseases is generally associated with more aggressive subtypes of disease.

In prostate cancer, both MYC and N-Myc have been shown to be amplified or overexpressed. Research suggests that MYC expression is an early event in prostate cancer that drives disease progression whereas N-Myc expression appears more in late-stage cancers and castrate-resistant prostate cancer, most often in neuroendocrine prostate cancer

(NEPC), an aggressive form of castrate-resistant prostate cancer. (348,349)Other settings

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where N-Myc and MYC have shown to both be expressed but in different subsets is small cell lung cancer. More remains to be learned about these settings but similar to prostate cancer, may drive different lineages or emerge at different stages of disease. (350,351)In summary, however, the literature points to a role for targeting N-MYC in more cancers than originally thought, both childhood and adult cancers.

N-Myc is regulated in a similar pattern to MYC with some exceptions.

Phosphorylation at S62 is a stabilizing event and phosphorylation at T58 initiates the process towards degradation. PP2A dephosphorylates N-Myc at S62 as it does MYC to enable identification by one of three E3 ligases (FBXW7, HUWE1, and TRUSS). The identification of kinases involved in this process are not all known, but in the case of neuroblastoma, it has been shown that CDK1 phosphorylates it at S62 and GSK3b is the kinase that phosphorylates it at T58(352). However, what may set N-Myc apart from MYC is that is has been well characterized to be further stabilized by an interaction with Aurora

Kinase A. Aurora Kinase A binds to MYC overlapping the phosphodegron and competing with its E3 ligase FBXW7 to further stabilize N-Myc. In many cancers where N-Myc is amplified or overexpressed, Aurora Kinase A is also amplified or overexpressed. Given the similarities, and even including the differences, between MYC and N-Myc, it can be hypothesized that SMAPs may have activity in cancers with N-Myc amplification or overexpression. Furthermore, given the stabilizing interaction between Aurora kinase A and N-Myc, it would be interesting to see if there is synergy with Aurora kinase inhibitors and SMAPs.(349,352-354) It is unclear if active PP2A could access S62 if N-Myc is protected by its interaction with Aurora Kinase A, therefore the use of an Aurora Kinase inhibitor may be necessary or it could be that the two approaches combined have synergy.

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Lastly, the work in KRAS mutant NSCLC demonstrates that an advantage of activating PP2A is that it has the potential to negatively regulate multiple signaling pathways driven by oncogenes. Not only do SMAPs degrade MYC in NSCLC but other published research demonstrates how effective SMAPs are at inhibiting MEK-ERK signaling. N-Myc stability is indirectly regulated through PI3K signaling. AKT phosphorylates and inhibits GSK3B which results in further stabilization of N-Myc.

(352,355)Thus, SMAPs could effectively contribute a 1-2 hit in a setting of PI3K activity along with N-Myc amplification or expression.

L-MYC

MYCL was first identified in small cell lung cancer. Of the three MYC family transcription factors, this is perhaps the least well described. Like N-Myc it has been shown that its expression is generally restricted to early stages of development and to certain tissue lineages. Its oncogenic potential has been validated in SCLC and it has been posited as a potential therapeutic target in SCLC. SCLC accounts for about 10-15% of all lung cancers.

It is fast-growing and aggressive and has few treatment options. While it's regulation has not been as well described, it can be hypothesized based on similarities in protein homology in all three transcription factors and their function that its stability may be similarly negatively regulated by PP2A in its phosphodegron. (231,356)Moreover, within SCLC, subsets emerge all driven by one of the MYC family members. Thus, regardless of which

MYC family transcription factor is driving tumor progression, SMAPs may be a viable treatment option for this disease in the absence of other viable targeted or effective approaches.

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3.3 PP2A, MYC, and CIP2A

The endogenous inhibitor of PP2A, CIP2A is well characterized for its inhibition of PP2A activity directed towards MYC. Recently, it was shown that this may be because

CIP2A imposes its inhibition through binding to the regulatory subunit B56a(357), the same subunit that is associated with modulation of MYC by PP2A. CIP2A is also shown to be upregulated in many cancers, often concurrently with MYC.(358) Moreover, research from our lab under review, demonstrates that SMAP binding to the PP2A holoenzyme is specific for certain holoenzymes. Specifically, it's shown that the SMAP, DT 061 (SMAP

1) stabilizes the holoenzyme containing the B56a subunit. Therefore, in the context of the work presented here, it is imperative to better understand how CIP2A may affect SMAP activity. CIP2A expression could inhibit SMAP 1 from binding or perhaps SMAP 1 can bind and promote assembly of the holoenzyme regardless of CIP2A expression.

Unpublished research in vitro demonstrates that SMAP 1 may successfully result in holoenzyme formation regardless of CIP2A expression. Upon treatment with SMAPs in a panel of triple negative breast cancer cell lines in vitro, CIP2A expression is decreased – in some cell lines it is undetectable within 24 hours while in others it is reduced by about

50% at the mRNA and protein level. Additionally, using genetic tools to knock down or overexpress CIP2A and gauge response to SMAP 1 as well as changes to MYC it seems that CIP2A expression does not change response so SMAPs. In the setting of CIP2A overexpression, changes to cell viability upon treatment with SMAP 1 was similar to the parental cell lines. Moreover, it did not change the degree to which MYC expression is decreased in the presence of SMAP 1. Conversely, when CIP2A is knocked down, the

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response to SMAPs also stayed the same. However, this research has not been rigorously carried out in the in vivo setting which may be a better setting to assess the consequences of CIP2A on SMAP anti-tumorigenic activity. Preliminary data in a TNBC cell selected for its dependency on a CIP2A gene signature was sensitive to SMAP 1 in vitro but was resistant to SMAP 1 treatment in vivo. This data is not exhaustive enough to demonstrate that CIP2A expression is driving resistance but it does provide a foundation that more inquiry into this area is warranted. CIP2A is overexpressed in triple-negative breast cancer, one of the settings that this work is based on. Thus as research moves forward, positioning

PP2A activation for the treatment of MYC driven cancers, this is an important area to investigate further as CIP2A overexpression contributes to the stability of MYC in many cancers. Specifically, in models of triple-negative breast cancer to start, we could knock out or overexpress CIP2A in cell lines that are known to grow in vivo and gauge changes to response based on CIP2A expression.

In addition to whether or not CIP2A expression could inhibit SMAP activity,

SMAPs may provide us with a better tool to study how CIP2A inhibits PP2A activity and if this inhibition is specific for certain holoenzymes or substrates. While research has shown that it binds to the B56a subunit, it does not rule out the possibility for other subunits. Furthermore, CIP2A has been shown to potentially impede PP2A mediated activity on AKT and DNA-PK(116,358,359). SMAPs could be a useful tool in the context of CIP2A overexpression to better understand holoenzymes inhibited by PP2A and the substrates its protecting. To start, assays such as co-immunoprecipitation assays, could be done to initially observe interactions between CIP2A and B subunits in the absence and

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presence of SMAPs. This information may be used to further parse out which substrates are affected by CIP2A and if SMAPs overcome this protection.

3.4 Understanding vulnerabilities in PP2A inactivated/MYC driven cancers

This research highlights the benefit of activating a tumor suppressor to inhibit an oncogene, hitting a cancer cell through at least two dependencies, possibly more if a cancer cell is dependent on multiple oncogenes that PP2A negatively regulates as exemplified in the models of KRAS driven NSCLC that are also dependent on MYC. However, the research does not address what cellular functions are affected the most by this strategy or what phenotype is targeted that drives the biological response observed. Further research into understanding the vulnerabilities most affected by PP2A activation may provide a better understanding to settings in which SMAPs may perform better as well as understanding better the cellular process that may be affected by PP2A activation and MYC inhibition. It remains to be seen if they are together targeting the same vulnerabilities, or if PP2A activation contributes to the negative regulation of one process independent of its effect on MYC. If the latter, this could cooperate with the consequence of MYC inhibition to drive the potent biology demonstrated in the research presented here.

MYC is associated with a variety of cellular processes that contribute to its oncogenic output. It drives growth through metabolic reprogramming, proliferation, negative regulation of apoptosis, among other processes. While the net sum of this output is cancer cell growth and oncogenesis, more recent research has suggested that some of the processes that MYC reprograms may be more critical to the success of the cancer cell than others. (222,223,280,328,360-363)For example, cells can become highly dependent on

MYC directed metabolic reprogramming such that a proposed approach to target MYC

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driven cells is to target their metabolic vulnerabilities. Understanding what vulnerabilities to target in MYC driven cells is important as you consider resistance and the chance for a cell to overcome one selective pressure or another. As MYC is a transcription factor, there remains the chance for cells to reprogram themselves an compensate for the loss of MYC.

It is hypothesized that the more efficiently we can target the cell's vulnerabilities, the less opportunity we then give to the cells to overcome the selective pressure.

Likewise, in the context of PP2A inhibition, cancer cells may become dependent on certain pathways and functions that make them vulnerable to its reactivation. The majority of research with SMAPs focuses on substrate identification and the consequences of inhibiting that substrate. But given its propensity to negatively regulate several oncogenic substrates and pathways, it is also a worthwhile endeavor to more broadly identify functional vulnerabilities in the cell. While this research demonstrates the potential for PP2A activation as a monotherapy, understanding a cell's vulnerabilities enables the development of rational combination therapies. This could result in a potent cancer effect and but also potentially get ahead of possible mechanisms of resistance that may develop upon PP2A activation. Conversely, instead of vulnerabilities, understanding which functional outputs remain unchanged in the context of PP2A activation could also help with rational combinations- designing combinations that target different functions within the cell would ideally minimize the chance for a cell to overcome a single selective pressure. Therefore, in addition to observing changes to cellular viability and tumor growth inhibition, a variety of screens in MYC driven cancers with SMAPs to study proliferation, metabolism, apoptosis, and autophagy could begin to predict what vulnerabilities are most likely responsible for the phenotype observed so far as well as which ones aren’t. This

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information may provide further insight into how PP2A exerts its tumor suppresive function as well as highlight areas to target with combination therapies. Lastly, given the diversity of tissue types where MYC is found, it would be best to continue these studies in a diverse set of cancer lines or models. It is likely the context dictates how and why the cell responses to PP2A activation and that its different across tissue types

3.5 Status of the PP2A- B56a Holoenzyme

As described, this work converges with research under review that demonstrates specificity for SMAP 1 for the B56a PP2A holoenzyme. This research supports our research in that PP2A- B56a is characterized as the holoenzyme that negatively regulates

MYC. But if SMAP mediated activation of PP2A is highly dependent on the stability of this holoenzyme, it would behoove us to further investigate how B56a is regulated in the cell as well as if it has any defined mutational burden in cancer. It may be that inhibition by CIP2A is the primary way in which the PP2A B56a containing holoenzyme is affected in cancer cells. But research suggests that the B subunits may be regulated through post- translational mechanisms that are not yet well understood and also acquire mutations in cancer(60,78,300,302,364). If this holoenzyme is in fact responsible for the bulk of the

SMAP directed activity against MYC, it will be important to understand if SMAPs can still assemble the holoenzyme in the context of dysregulated B56a and if dysregulation may alter the output of the subunit, how that might change the response to SMAPs in MYC driven cancers.

3.6 Understanding mechanisms of resistance to SMAPs in MYC driven cancers

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The ability to target MYC driven cancers is of immense benefit to future patients.

However, as this approach to target MYC with SMAPs is developed, it would be of great benefit to understand if and how resistance may develop to SMAPs. As effective as this approach is in some models of MYC driven cancers it would be naïve to assume that cancers won’t find ways to overcome this selective pressure. Cancer cells are driven to survive and often evolve ways to overcome treatment strategies in patients. Indeed, some of the models presented within this work illuminate this point as some SMAP treatment tumors appear to start growing again the longer they are exposed to SMAPs. Garnering a better understanding of how these tumors develop resistance is of great advantage to the success of this strategy. Understanding resistance enables the design of combination therapies that may pack a one, two punch reducing the cells ability to acquire resistance or knowing the mechanism of resistance can help identify a second line therapy that targets the mechanism driving resistance. Alternatively, understanding how resistance develops enables the identification of subsets of patients with MYC driven cancers that may be more sensitive or resistant to this approach from the beginning.

There are multiple approaches to identifying mechanisms of resistance to targeted therapies described in the literature.(365) A notable approach in vitro is the exposure of cells to increasing concentrations of a proposed treatment strategy over time. This setting hypothetically enables cells to acquire mechanisms of resistance that can be identified when the cells are resistant to high relative concentrations of the stressor they’re being exposed to. However, unpublished efforts in the Narla lab did not yield a resistant population of cells to be studied further.

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A second effort in the lab using a forward genetics approach, that is the creation of random mutations in populations of cells was also used as a means of understanding resistance to SMAPs. In this approach if a population of cells emerged resistant, the technology used, VBIM(366), would enable us to backtrack and identify the mutation that may be driving resistance in these cells. Unfortunately, these efforts did not yield any resistant populations as well.

Some of the in vivo models presented within this work highlight an approach that may yield resistance. In the Daudi and MDA-MB-231 models, the slope of the mean tumor volume of mice treated with SMAPs begins to increase at the end suggesting that at least some of the tumors within these groups may be becoming resistant to SMAPs over time. These models are a great place to start in delineating resistance. If they could be repeated, allowing the SMAP treated tumors to continue growing past the day when these studies were terminated, we may be better to fully assess if resistance is growing and then go back and try to assess why or how these tumors acquire resistance to SMAPs. A proposed study would be to have a short Pd arm that looks at changes to MYC within a few days of exposure to SMAPs, an arm similar to the ones done so far where at the end of the study we see about a 50% decrease in MYC and then an arm taken out for resistance.

These arms could be compared at the end to look for sensitivity in regards to how MYC is degraded over time. This will allow us to discern if the resistance itself is through MYC.

Another candidate approach to identify a resistance mechanism would be to assess whether the PP2A B56a holoenzyme is stabilized in the presence of SMAPs to the same degree at the beginning and middle of study as well as in the arm taken out to when resistance develops. Perhaps holoenzyme formation is changed due to acquired mutations or changes

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in expression in one of the individual subunits. If neither of these candidate approaches yields a discernible mechanism, the tumors may need to be assessed more globally to identify changes that may be contributing to resistance.

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References

1. Brautigan, D. L. (2013) Protein Ser/Thr phosphatases--the ugly ducklings of cell signalling. Febs J 280, 324-345 2. Shi, Y. (2009) Serine/threonine phosphatases: mechanism through structure. Cell 139, 468-484 3. Stone, S. R., Hofsteenge, J., and Hemmings, B. A. (1987) Molecular cloning of cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A. Biochemistry 26, 7215-7220 4. Khew-Goodall, Y., and Hemmings, B. A. (1988) Tissue-specific expression of mRNAs encoding alpha- and beta-catalytic subunits of protein phosphatase 2A. FEBS Lett 238, 265-268 5. Eichhorn, P. J., Creyghton, M. P., and Bernards, R. (2009) Protein phosphatase 2A regulatory subunits and cancer. Biochim Biophys Acta 1795, 1-15 6. Andrade, M. A., and Bork, P. (1995) HEAT repeats in the Huntington's disease protein. Nat Genet 11, 115-116 7. Groves, M. R., Hanlon, N., Turowski, P., Hemmings, B. A., and Barford, D. (1999) The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96, 99-110 8. Wozniak, E., Oldziej, S., and Ciarkowski, J. (2000) Molecular modeling of the catalytic domain of serine/threonine phosphatase-1 with the Zn2+ and Mn2+ di- nuclear ion centers in the active site. Comput Chem 24, 381-390 9. Xing, Y., Xu, Y., Chen, Y., Jeffrey, P. D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J. B., and Shi, Y. (2006) Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins. Cell 127, 341-353 10. Longin, S., Zwaenepoel, K., Louis, J. V., Dilworth, S., Goris, J., and Janssens, V. (2007) Selection of protein phosphatase 2A regulatory subunits is mediated by the C terminus of the catalytic Subunit. J Biol Chem 282, 26971-26980 11. Kamibayashi, C., Lickteig, R. L., Estes, R., Walter, G., and Mumby, M. C. (1992) Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits. J Biol Chem 267, 21864- 21872 12. McCright, B., Rivers, A. M., Audlin, S., and Virshup, D. M. (1996) The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation- induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271, 22081-22089 13. Cho, U. S., and Xu, W. (2007) Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53-57 14. Wlodarchak, N., Guo, F., Satyshur, K. A., Jiang, L., Jeffrey, P. D., Sun, T., Stanevich, V., Mumby, M. C., and Xing, Y. (2013) Structure of the Ca2+- dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation. Cell Res 23, 931-946 15. Gentry, M. S., and Hallberg, R. L. (2002) Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle. Mol Biol Cell 13, 3477-3492

111

16. Ghaemmaghami, S., Huh, W. K., Bower, K., Howson, R. W., Belle, A., Dephoure, N., O'Shea, E. K., and Weissman, J. S. (2003) Global analysis of protein expression in yeast. Nature 425, 737-741 17. Sents, W., Ivanova, E., Lambrecht, C., Haesen, D., and Janssens, V. (2013) The biogenesis of active protein phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity. Febs J 280, 644-661 18. Beck, M., Schmidt, A., Malmstroem, J., Claassen, M., Ori, A., Szymborska, A., Herzog, F., Rinner, O., Ellenberg, J., and Aebersold, R. (2011) The quantitative proteome of a human cell line. Mol Syst Biol 7, 549 19. Li, X., Scuderi, A., Letsou, A., and Virshup, D. M. (2002) B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster. Mol Cell Biol 22, 3674-3684 20. Silverstein, A. M., Barrow, C. A., Davis, A. J., and Mumby, M. C. (2002) Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits. Proc Natl Acad Sci U S A 99, 4221-4226 21. Strack, S., Cribbs, J. T., and Gomez, L. (2004) Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival. J Biol Chem 279, 47732-47739 22. Janssens, V., Jordens, J., Stevens, I., Van Hoof, C., Martens, E., De Smedt, H., Engelborghs, Y., Waelkens, E., and Goris, J. (2003) Identification and functional analysis of two Ca2+-binding EF-hand motifs in the B"/PR72 subunit of protein phosphatase 2A. J Biol Chem 278, 10697-10706 23. Chen, W., Arroyo, J. D., Timmons, J. C., Possemato, R., and Hahn, W. C. (2005) Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity. Cancer Res 65, 8183-8192 24. Liu, F., Grundke-Iqbal, I., Iqbal, K., and Gong, C. X. (2005) Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation. Eur J Neurosci 22, 1942-1950 25. Gong, C. X., Singh, T. J., Grundke-Iqbal, I., and Iqbal, K. (1993) Phosphoprotein phosphatase activities in Alzheimer disease brain. J Neurochem 61, 921-927 26. Vogelsberg-Ragaglia, V., Schuck, T., Trojanowski, J. Q., and Lee, V. M. (2001) PP2A mRNA expression is quantitatively decreased in Alzheimer's disease hippocampus. Exp Neurol 168, 402-412 27. Sontag, E., Luangpirom, A., Hladik, C., Mudrak, I., Ogris, E., Speciale, S., and White, C. L., 3rd. (2004) Altered expression levels of the protein phosphatase 2A ABalphaC enzyme are associated with Alzheimer disease pathology. J Neuropathol Exp Neurol 63, 287-301 28. Gong, C. X., Shaikh, S., Wang, J. Z., Zaidi, T., Grundke-Iqbal, I., and Iqbal, K. (1995) Phosphatase activity toward abnormally phosphorylated tau: decrease in Alzheimer disease brain. J Neurochem 65, 732-738 29. Liu, R., Zhou, X. W., Tanila, H., Bjorkdahl, C., Wang, J. Z., Guan, Z. Z., Cao, Y., Gustafsson, J. A., Winblad, B., and Pei, J. J. (2008) Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology. J Cell Mol Med 12, 241-257 30. Voronkov, M., Braithwaite, S. P., and Stock, J. B. (2011) Phosphoprotein phosphatase 2A: a novel druggable target for Alzheimer's disease. Future Med Chem 3, 821-833

112

31. Tian, Q., and Wang, J. (2002) Role of serine/threonine protein phosphatase in Alzheimer's disease. Neurosignals 11, 262-269 32. Begum, N., and Ragolia, L. (1996) cAMP counter-regulates insulin-mediated protein phosphatase-2A inactivation in rat skeletal muscle cells. J Biol Chem 271, 31166-31171 33. Hojlund, K., Poulsen, M., Staehr, P., Brusgaard, K., and Beck-Nielsen, H. (2002) Effect of insulin on protein phosphatase 2A expression in muscle in type 2 diabetes. Eur J Clin Invest 32, 918-923 34. Lipina, C., and Hundal, H. S. (2014) Carnosic acid stimulates glucose uptake in skeletal muscle cells via a PME-1/PP2A/PKB signalling axis. Cell Signal 26, 2343- 2349 35. Wallace, A. M., Hardigan, A., Geraghty, P., Salim, S., Gaffney, A., Thankachen, J., Arellanos, L., D'Armiento, J. M., and Foronjy, R. F. (2012) Protein phosphatase 2A regulates innate immune and proteolytic responses to cigarette smoke exposure in the lung. Toxicol Sci 126, 589-599 36. Kirchhefer, U., Brekle, C., Eskandar, J., Isensee, G., Kucerova, D., Muller, F. U., Pinet, F., Schulte, J. S., Seidl, M. D., and Boknik, P. (2014) Cardiac function is regulated by B56alpha-mediated targeting of protein phosphatase 2A (PP2A) to contractile relevant substrates. J Biol Chem 289, 33862-33873 37. Perrotti, D., and Neviani, P. (2013) Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol 14, e229-238 38. Sablina, A. A., and Hahn, W. C. (2008) SV40 small T antigen and PP2A phosphatase in cell transformation. Cancer Metastasis Rev 27, 137-146 39. Ruediger, R., Ruiz, J., and Walter, G. (2011) Human cancer-associated mutations in the Aalpha subunit of protein phosphatase 2A increase lung cancer incidence in Aalpha knock-in and knockout mice. Mol Cell Biol 31, 3832-3844 40. Ruediger, R., Pham, H. T., and Walter, G. (2001) Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene 20, 1892-1899 41. Calin, G. A., di Iasio, M. G., Caprini, E., Vorechovsky, I., Natali, P. G., Sozzi, G., Croce, C. M., Barbanti-Brodano, G., Russo, G., and Negrini, M. (2000) Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms. Oncogene 19, 1191-1195 42. Ruediger, R., Pham, H. T., and Walter, G. (2001) Disruption of protein phosphatase 2A subunit interaction in human cancers with mutations in the A alpha subunit gene. Oncogene 20, 10-15 43. McConechy, M. K., Anglesio, M. S., Kalloger, S. E., Yang, W., Senz, J., Chow, C., Heravi-Moussavi, A., Morin, G. B., Mes-Masson, A. M., Carey, M. S., McAlpine, J. N., Kwon, J. S., Prentice, L. M., Boyd, N., Shah, S. P., Gilks, C. B., and Huntsman, D. G. (2011) Subtype-specific mutation of PPP2R1A in endometrial and ovarian carcinomas. J Pathol 223, 567-573 44. Shih Ie, M., and Wang, T. L. (2011) Mutation of PPP2R1A: a new clue in unveiling the pathogenesis of uterine serous carcinoma. J Pathol 224, 1-4 45. Seshagiri, S., Stawiski, E. W., Durinck, S., Modrusan, Z., Storm, E. E., Conboy, C. B., Chaudhuri, S., Guan, Y., Janakiraman, V., Jaiswal, B. S., Guillory, J., Ha, C.,

113

Dijkgraaf, G. J., Stinson, J., Gnad, F., Huntley, M. A., Degenhardt, J. D., Haverty, P. M., Bourgon, R., Wang, W., Koeppen, H., Gentleman, R., Starr, T. K., Zhang, Z., Largaespada, D. A., Wu, T. D., and de Sauvage, F. J. (2012) Recurrent R- spondin fusions in colon cancer. Nature 488, 660-664 46. Jones, S., Wang, T. L., Shih Ie, M., Mao, T. L., Nakayama, K., Roden, R., Glas, R., Slamon, D., Diaz, L. A., Jr., Vogelstein, B., Kinzler, K. W., Velculescu, V. E., and Papadopoulos, N. (2010) Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science 330, 228-231 47. Nagendra, D. C., Burke, J., 3rd, Maxwell, G. L., and Risinger, J. I. (2012) PPP2R1A mutations are common in the serous type of endometrial cancer. Mol Carcinog 51, 826-831 48. Colella, S., Ohgaki, H., Ruediger, R., Yang, F., Nakamura, M., Fujisawa, H., Kleihues, P., and Walter, G. (2001) Reduced expression of the Aalpha subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aalpha and Abeta subunit genes. Int J Cancer 93, 798-804 49. Baysal, B. E., Farr, J. E., Goss, J. R., Devlin, B., and Richard, C. W., 3rd. (1998) Genomic organization and precise physical location of protein phosphatase 2A regulatory subunit A beta isoform gene on chromosome band 11q23. Gene 217, 107-116 50. Kiely, M., and Kiely, P. A. (2015) PP2A: The Wolf in Sheep's Clothing? Cancers (Basel) 7, 648-669 51. Wang, S. S., Esplin, E. D., Li, J. L., Huang, L., Gazdar, A., Minna, J., and Evans, G. A. (1998) Alterations of the PPP2R1B gene in human lung and colon cancer. Science 282, 284-287 52. Sablina, A. A., Chen, W., Arroyo, J. D., Corral, L., Hector, M., Bulmer, S. E., DeCaprio, J. A., and Hahn, W. C. (2007) The tumor suppressor PP2A Abeta regulates the RalA GTPase. Cell 129, 969-982 53. Kalla, C., Scheuermann, M. O., Kube, I., Schlotter, M., Mertens, D., Dohner, H., Stilgenbauer, S., and Lichter, P. (2007) Analysis of 11q22-q23 deletion target genes in B-cell chronic lymphocytic leukaemia: evidence for a pathogenic role of NPAT, CUL5, and PPP2R1B. Eur J Cancer 43, 1328-1335 54. Chou, H.-C., Chen, C.-H., Lee, H.-S., Lee, C.-Z., Huang, G.-T., Yang, P.-M., Lee, P.-H., and Sheu, J.-C. (2007) Alterations of tumour suppressor PP2R1B in hepatocellular carcinoma. Cancer Lett 253, 138-143 55. Hemmings, B. A., Adams-Pearson, C., Maurer, F., Muller, P., Goris, J., Merlevede, W., Hofsteenge, J., and Stone, S. R. (1990) alpha- and beta-forms of the 65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating structure. Biochemistry 29, 3166-3173 56. Shouse, G. P., Nobumori, Y., and Liu, X. (2010) A B56gamma mutation in lung cancer disrupts the p53-dependent tumor-suppressor function of protein phosphatase 2A. Oncogene 29, 3933-3941 57. Cheng, Y., Liu, W., Kim, S. T., Sun, J., Lu, L., Zheng, S. L., Isaacs, W. B., and Xu, J. (2011) Evaluation of PPP2R2A as a prostate cancer susceptibility gene: a comprehensive germline and somatic study. Cancer Genet 204, 375-381 58. Curtis, C., Shah, S. P., Chin, S. F., Turashvili, G., Rueda, O. M., Dunning, M. J., Speed, D., Lynch, A. G., Samarajiwa, S., Yuan, Y., Graf, S., Ha, G., Haffari, G.,

114

Bashashati, A., Russell, R., McKinney, S., Langerod, A., Green, A., Provenzano, E., Wishart, G., Pinder, S., Watson, P., Markowetz, F., Murphy, L., Ellis, I., Purushotham, A., Borresen-Dale, A. L., Brenton, J. D., Tavare, S., Caldas, C., and Aparicio, S. (2012) The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346-352 59. Mosca, L., Musto, P., Todoerti, K., Barbieri, M., Agnelli, L., Fabris, S., Tuana, G., Lionetti, M., Bonaparte, E., Sirchia, S. M., Grieco, V., Bianchino, G., D'Auria, F., Statuto, T., Mazzoccoli, C., De Luca, L., Petrucci, M. T., Morabito, F., Offidani, M., Di Raimondo, F., Falcone, A., Caravita, T., Omede, P., Boccadoro, M., Palumbo, A., and Neri, A. (2013) Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with changes in transcriptional profiles. Am J Hematol 88, 16-23 60. Ito, A., Kataoka, T. R., Watanabe, M., Nishiyama, K., Mazaki, Y., Sabe, H., Kitamura, Y., and Nojima, H. (2000) A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. The EMBO journal 19, 562-571 61. Mannava, S., Omilian, A. R., Wawrzyniak, J. A., Fink, E. E., Zhuang, D., Miecznikowski, J. C., Marshall, J. R., Soengas, M. S., Sears, R. C., Morrison, C. D., and Nikiforov, M. A. (2012) PP2A-B56alpha controls oncogene-induced senescence in normal and tumor human melanocytic cells. Oncogene 31, 1484- 1492 62. Deichmann, M., Thome, M., Benner, A., Egner, U., Hartschuh, W., and Naher, H. (2002) PTEN/MMAC1 expression in melanoma resection specimens. Br J Cancer 87, 1431-1436 63. Spencer, E. S., Bluemn, E. G., Johnston, R., Zhang, X., Gordon, R. R., Lewinshtein, D., Lucas, J., Nelson, P., and Porter, C. R. (2012) Association of decreased expression of protein phosphatase 2A subunit PR55γ (PPP2R2C) with an increased risk of metastases and prostate cancer-specific mortality. Journal of Clinical Oncology 30 64. Ruvolo, P. P., Qui, Y. H., Coombes, K. R., Zhang, N., Ruvolo, V. R., Borthakur, G., Konopleva, M., Andreeff, M., and Kornblau, S. M. (2011) Low expression of PP2A regulatory subunit B55alpha is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients. Leukemia 25, 1711-1717 65. Muggerud, A. A., Ronneberg, J. A., Warnberg, F., Botling, J., Busato, F., Jovanovic, J., Solvang, H., Bukholm, I., Borresen-Dale, A. L., Kristensen, V. N., Sorlie, T., and Tost, J. (2010) Frequent aberrant DNA methylation of ABCB1, FOXC1, PPP2R2B and PTEN in ductal carcinoma in situ and early invasive breast cancer. Breast cancer research : BCR 12, R3 66. Tan, J., Lee, P. L., Li, Z., Jiang, X., Lim, Y. C., Hooi, S. C., and Yu, Q. (2010) B55beta-associated PP2A complex controls PDK1-directed myc signaling and modulates rapamycin sensitivity in colorectal cancer. Cancer Cell 18, 459-471 67. Bhardwaj, A., Singh, S., Srivastava, S. K., Honkanen, R. E., Reed, E., and Singh, A. P. (2011) Modulation of protein phosphatase 2A activity alters androgen- independent growth of prostate cancer cells: therapeutic implications. Mol Cancer Ther 10, 720-731

115

68. Singh, A. P., Bafna, S., Chaudhary, K., Venkatraman, G., Smith, L., Eudy, J. D., Johansson, S. L., Lin, M. F., and Batra, S. K. (2008) Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgen-independent prostate cancer cells. Cancer Lett 259, 28-38 69. Ramaswamy, K., Spitzer, B., and Kentsis, A. (2015) Therapeutic Re-Activation of Protein Phosphatase 2A in Acute Myeloid Leukemia. Front Oncol 5, 16 70. Sallman, D. A., Wei, S., and List, A. (2014) PP2A: The Achilles Heal in MDS with 5q Deletion. Front Oncol 4, 264 71. De Baere, I., Derua, R., Janssens, V., Van Hoof, C., Waelkens, E., Merlevede, W., and Goris, J. (1999) Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue. Biochemistry 38, 16539-16547 72. Ogris, E., Du, X., Nelson, K. C., Mak, E. K., Yu, X. X., Lane, W. S., and Pallas, D. C. (1999) A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A. J Biol Chem 274, 14382-14391 73. Xing, Y. N., Li, Z., Chen, Y., Stock, J. B., Jeffrey, P. D., and Shi, Y. G. (2008) Structural mechanism of demethylation and inactivation of protein phosphatase 2A. Cell 133, 154-163 74. Ortega-Gutierrez, S., Leung, D., Ficarro, S., Peters, E. C., and Cravatt, B. F. (2008) Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. Plos One 3, e2486 75. Lee, J. A., and Pallas, D. C. (2007) Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells. J Biol Chem 282, 30974-30984 76. Xia, X., Gholkar, A., Senese, S., and Torres, J. Z. (2015) A LCMT1-PME-1 Methylation Equilibrium Controls Mitotic Spindle Size. Cell Cycle, 0 77. Ikehara, T., Ikehara, S., Imamura, S., Shinjo, F., and Yasumoto, T. (2007) Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B). Biochem Bioph Res Co 354, 1052-1057 78. Janssens, V., Longin, S., and Goris, J. (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends Biochem Sci 33, 113-121 79. Gentry, M. S., Li, Y., Wei, H., Syed, F. F., Patel, S. H., Hallberg, R. L., and Pallas, D. C. (2005) A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers. Eukaryot Cell 4, 1029-1040 80. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J. B., and Broach, J. R. (2000) Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J 19, 5672-5681 81. Yu, X. X., Du, X., Moreno, C. S., Green, R. E., Ogris, E., Feng, Q., Chou, L., McQuoid, M. J., and Pallas, D. C. (2001) Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but

116

not SG2NA, striatin, or polyomavirus middle tumor antigen. Mol Biol Cell 12, 185- 199 82. Xu, Y., Chen, Y., Zhang, P., Jeffrey, P. D., and Shi, Y. (2008) Structure of a protein phosphatase 2A holoenzyme: insights into B55-mediated Tau dephosphorylation. Mol Cell 31, 873-885 83. Sontag, J. M., Nunbhakdi-Craig, V., Montgomery, L., Arning, E., Bottiglieri, T., and Sontag, E. (2008) Folate deficiency induces in vitro and mouse brain region- specific downregulation of leucine carboxyl methyltransferase-1 and protein phosphatase 2A B(alpha) subunit expression that correlate with enhanced tau phosphorylation. J Neurosci 28, 11477-11487 84. Sontag, J. M., Nunbhakdi-Craig, V., Mitterhuber, M., Ogris, E., and Sontag, E. (2010) Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells. J Neurochem 115, 1455-1465 85. Sontag, J. M., Nunbhakdi-Craig, V., and Sontag, E. (2013) Leucine carboxyl methyltransferase 1 (LCMT1)-dependent methylation regulates the association of protein phosphatase 2A and Tau protein with plasma membrane microdomains in neuroblastoma cells. J Biol Chem 288, 27396-27405 86. Yang, C. C., Kuai, X. X., Li, Y. L., Zhang, L., Yu, J. C., and Li, L. (2013) Cornel Iridoid Glycoside Attenuates Tau Hyperphosphorylation by Inhibition of PP2A Demethylation. Evid Based Complement Alternat Med 2013, 108486 87. Longin, S., Jordens, J., Martens, E., Stevens, I., Janssens, V., Rondelez, E., De Baere, I., Derua, R., Waelkens, E., Goris, J., and Van Hoof, C. (2004) An inactive protein phosphatase 2A population is associated with methylesterase and can be re- activated by the phosphotyrosyl phosphatase activator. The Biochemical journal 380, 111-119 88. Fellner, T., Lackner, D. H., Hombauer, H., Piribauer, P., Mudrak, I., Zaragoza, K., Juno, C., and Ogris, E. (2003) A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo. Genes Dev 17, 2138-2150 89. Jordens, J., Janssens, V., Longin, S., Stevens, I., Martens, E., Bultynck, G., Engelborghs, Y., Lescrinier, E., Waelkens, E., Goris, J., and Van Hoof, C. (2006) The protein phosphatase 2A phosphatase activator is a novel peptidyl-prolyl cis/trans-isomerase. J Biol Chem 281, 6349-6357 90. Leulliot, N., Vicentini, G., Jordens, J., Quevillon-Cheruel, S., Schiltz, M., Barford, D., van Tilbeurgh, H., and Goris, J. (2006) Crystal structure of the PP2A phosphatase activator: implications for its PP2A-specific PPIase activity. Mol Cell 23, 413-424 91. Kong, M., Fox, C. J., Mu, J., Solt, L., Xu, A., Cinalli, R. M., Birnbaum, M. J., Lindsten, T., and Thompson, C. B. (2004) The PP2A-associated protein alpha4 is an essential inhibitor of apoptosis. Science 306, 695-698 92. LeNoue-Newton, M., Watkins, G. R., Zou, P., Germane, K. L., McCorvey, L. R., Wadzinski, B. E., and Spiller, B. W. (2011) The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation. J Biol Chem 286, 17665-17671

117

93. Li, M., Guo, H., and Damuni, Z. (1995) Purification and characterization of two potent heat-stable protein inhibitors of protein phosphatase 2A from bovine kidney. Biochemistry 34, 1988-1996 94. Li, M., Makkinje, A., and Damuni, Z. (1996) The myeloid leukemia-associated protein SET is a potent inhibitor of protein phosphatase 2A. Journal of Biological Chemistry 271, 11059-11062 95. Habrukowich, C., Han, D. K., Le, A., Rezaul, K., Pan, W., Ghosh, M., Li, Z. G., Dodge-Kafka, K., Jiang, X. J., Bittman, R., and Hla, T. (2010) Sphingosine Interaction with Acidic Leucine-rich Nuclear Phosphoprotein-32A (ANP32A) Regulates PP2A Activity and Cyclooxygenase (COX)-2 Expression in Human Endothelial Cells. Journal of Biological Chemistry 285, 26825-26831 96. Gharbi-Ayachi, A., Labbe, J. C., Burgess, A., Vigneron, S., Strub, J. M., Brioudes, E., Van-Dorsselaer, A., Castro, A., and Lorca, T. (2010) The Substrate of Greatwall Kinase, Arpp19, Controls Mitosis by Inhibiting Protein Phosphatase 2A. Science 330, 1673-1677 97. Mochida, S., Maslen, S. L., Skehel, M., and Hunt, T. (2010) Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science 330, 1670-1673 98. Kawabe, T., Muslin, A. J., and Korsmeyer, S. J. (1997) HOX11 interacts with protein phosphatases PP2A and PP1 and disrupts a G2/M cell-cycle checkpoint. Nature 385, 454-458 99. Saito, S., Miyaji-Yamaguchi, M., Shimoyama, T., and Nagata, K. (1999) Functional domains of template-activating factor-I as a protein phosphatase 2A inhibitor. Biochem Bioph Res Co 259, 471-475 100. Adachi, Y., Pavlakis, G. N., and Copeland, T. D. (1994) Identification and Characterization of Set, a Nuclear Phosphoprotein Encoded by the Translocation Break Point in Acute Undifferentiated Leukemia. Journal of Biological Chemistry 269, 2258-2262 101. Saito, S., Miyaji-Yamaguchi, M., and Nagata, K. (2004) Aberrant intracellular localization of set-can fusion protein, associated with a leukemia, disorganizes nuclear export. International Journal of Cancer 111, 501-507 102. Vonlindern, M., Vanbaal, S., Wiegant, J., Raap, A., Hagemeijer, A., and Grosveld, G. (1992) Can, a Putative Oncogene Associated with Myeloid Leukemogenesis, May Be Activated by Fusion of Its 3' 1/2 to Different Genes - Characterization of the Set Gene. Mol Cell Biol 12, 3346-3355 103. Christensen, D. J., Chen, Y. W., Oddo, J., Matta, K. M., Neil, J., Davis, E. D., Volkheimer, A. D., Lanasa, M. C., Friedman, D. R., Goodman, B. K., Gockerman, J. P., Diehl, L. F., de Castro, C. M., Moore, J. O., Vitek, M. P., and Weinberg, J. B. (2011) SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target. Blood 118, 4150-4158 104. Cristobal, I., Garcia-Orti, L., Cirauqui, C., Cortes-Lavaud, X., Garcia-Sanchez, M. A., Calasanz, M. J., and Odero, M. D. (2012) Overexpression of SET is a recurrent event associated with poor outcome and contributes to protein phosphatase 2A inhibition in acute myeloid leukemia. Haematol-Hematol J 97, 543-550

118

105. Neviani, P., Santhanam, R., Trotta, R., Notari, M., Blaser, B. W., Liu, S., Mao, H., Chang, J. S., Galietta, A., Uttam, A., Roy, D. C., Valtieri, M., Bruner-Klisovic, R., Caligiuri, M. A., Bloomfield, C. D., Marcucci, G., and Perrotti, D. (2005) The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer Cell 8, 355-368 106. Van Vlierberghe, P., van Grotel, M., Tchinda, J., Lee, C., Beverloo, H. B., van der Spek, P. J., Stubbs, A., Cools, J., Nagata, K., Fornerod, M., Buijs-Gladdines, J., Horstmann, M., van Wering, E. R., Soulier, J., Pieters, R., and Meijerink, J. P. P. (2008) The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia. Blood 111, 4668-4680 107. Liu, H., Gu, Y., Wang, H., Yin, J., Zheng, G., Zhang, Z., Lu, M., Wang, C., and He, Z. (2015) Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer. Oncotarget 6, 14913-14925 108. Janghorban, M., Farrell, A. S., Allen-Petersen, B. L., Pelz, C., Daniel, C. J., Oddo, J., Langer, E. M., Christensen, D. J., and Sears, R. C. (2014) Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer. Proc Natl Acad Sci U S A 111, 9157- 9162 109. Cristobal, I., Rincon, R., Manso, R., Carames, C., Zazo, S., Madoz-Gurpide, J., Rojo, F., and Garcia-Foncillas, J. (2015) Deregulation of the PP2A inhibitor SET shows promising therapeutic implications and determines poor clinical outcome in patients with metastatic colorectal cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 21, 347-356 110. Anazawa, Y., Nakagawa, H., Furihara, M., Ashida, S., Tamura, K., Yoshioka, H., Shuin, T., Fujioka, T., Katagiri, T., and Nakamura, Y. (2005) PCOTH, a novel gene overexpressed in prostate cancers, promotes prostate cancer cell growth through phosphorylation of oncoprotein TAF-l beta/SET. Cancer Res 65, 4578-4586 111. Irie, A., Harada, K., Araki, N., and Nishimura, Y. (2012) Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A. Plos One 7 112. Arnaud, L., Chen, S., Liu, F., Li, B., Khatoon, S., Grundke-Iqbal, I., and Iqbal, K. (2011) Mechanism of inhibition of PP2A activity and abnormal hyperphosphorylation of tau by I2(PP2A)/SET. FEBS Lett 585, 2653-2659 113. Yu, G., Yan, T., Feng, Y., Liu, X., Xia, Y., Luo, H., Wang, J. Z., and Wang, X. (2013) Ser9 phosphorylation causes cytoplasmic detention of I2PP2A/SET in Alzheimer disease. Neurobiol Aging 34, 1748-1758 114. Saito, S., Miyaji-Yamaguchi, M., Shimoyama, T., and Nagata, K. (1999) Functional domains of template-activating factor-I as a protein phosphatase 2A inhibitor. Biochem Bioph Res Co 259, 471-475 115. Laine, A., Sihto, H., Come, C., Rosenfeldt, M. T., Zwolinska, A., Niemela, M., Khanna, A., Chan, E. K., Kahari, V. M., Kellokumpu-Lehtinen, P. L., Sansom, O. J., Evan, G. I., Junttila, M. R., Ryan, K. M., Marine, J. C., Joensuu, H., and Westermarck, J. (2013) Senescence sensitivity of breast cancer cells is defined by positive feedback loop between CIP2A and E2F1. Cancer Discov 3, 182-197

119

116. Puustinen, P., Rytter, A., Mortensen, M., Kohonen, P., Moreira, J. M., and Jaattela, M. (2014) CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. J Cell Biol 204, 713-727 117. Guenebeaud, C., Goldschneider, D., Castets, M., Guix, C., Chazot, G., Delloye- Bourgeois, C., Eisenberg-Lerner, A., Shohat, G., Zhang, M., Laudet, V., Kimchi, A., Bernet, A., and Mehlen, P. (2010) The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase. Mol Cell 40, 863-876 118. Junttila, M. R., Puustinen, P., Niemela, M., Ahola, R., Arnold, H., Bottzauw, T., Ala-Aho, R., Nielsen, C., Ivaska, J., Taya, Y., Lu, S. L., Lin, S. J., Chan, E. K. L., Wang, X. J., Grenman, R., Kast, J., Kallunki, T., Sears, R., Kahari, V. M., and Westermarck, J. (2007) CIP2A inhibits PP2A in human malignancies. Cell 130, 51- 62 119. Bockelman, C., Koskensalo, S., Hagstrom, J., Lundin, M., Ristimaki, A., and Haglund, C. (2012) CIP2A overexpression is associated with c-Myc expression in colorectal cancer. Cancer Biol Ther 13, 289-295 120. Bockelman, C., Lassus, H., Hemmes, A., Leminen, A., Westermarck, J., Haglund, C., Butzow, R., and Ristimaki, A. (2011) Prognostic role of CIP2A expression in serous ovarian cancer. Br J Cancer 105, 989-995 121. Come, C., Laine, A., Chanrion, M., Edgren, H., Mattila, E., Liu, X. L., Jonkers, J., Ivaska, J., Isola, J., Darbon, J. M., Kallioniemi, O., Thezenas, S., and Westermarck, J. (2009) CIP2A Is Associated with Human Breast Cancer Aggressivity. Clin Cancer Res 15, 5092-5100 122. Dong, Q. Z., Wang, Y., Dong, X. J., Li, Z. X., Tang, Z. P., Cui, Q. Z., and Wang, E. H. (2011) CIP2A is Overexpressed in Non-Small Cell Lung Cancer and Correlates with Poor Prognosis. Ann Surg Oncol 18, 857-865 123. Fang, Y. Y., Li, Z. T., Wang, X. X., and Zhang, S. L. (2012) CIP2A is overexpressed in human ovarian cancer and regulates cell proliferation and apoptosis. Tumor Biol 33, 2299-2306 124. He, H., Wu, G., Li, W. J., Cao, Y. C., and Liu, Y. F. (2012) CIP2A Is Highly Expressed in Hepatocellular Carcinoma and Predicts Poor Prognosis. Diagn Mol Pathol 21, 143-149 125. Huang, L. P., Adelson, M. E., Mordechai, E., and Trama, J. P. (2010) CIP2A expression is elevated in cervical cancer. Cancer Biomark 8, 309-317 126. Huang, L. P., Savoly, D., Sidi, A. A., Adelson, M. E., Mordechai, E., and Trama, J. P. (2012) CIP2A protein expression in high-grade, high-stage bladder cancer. Cancer medicine 1, 76-81 127. Khanna, A., Bockelman, C., Hemmes, A., Junttila, M. R., Wiksten, J. P., Lundin, M., Junnila, S., Murphy, D. J., Evan, G. I., Haglund, C., Westermarck, J., and Ristimaki, A. (2009) MYC-Dependent Regulation and Prognostic Role of CIP2A in Gastric Cancer. J Natl Cancer I 101, 793-805 128. Kikuno, R., Nagase, T., Ishikawa, K., Hirosawa, M., Miyajima, N., Tanaka, A., Kotani, H., Nomura, N., and Ohara, O. (1999) Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA research : an

120

international journal for rapid publication of reports on genes and genomes 6, 197- 205 129. Li, W. J., Ge, Z., Liu, C., Liu, Z. F., Bjorkholm, M., Jia, J. H., and Xu, D. W. (2008) CIP2A is overexpressed in gastric cancer and its depletion leads to impaired clonogenicity, senescence, or differentiation of tumor cells. Clin Cancer Res 14, 3722-3728 130. Qu, W., Li, W. J., Wei, L., Xing, L. G., Wang, X. W., and Yu, J. M. (2012) CIP2A is overexpressed in esophageal squamous cell carcinoma. Med Oncol 29, 113-118 131. Rantanen, T., Kauttu, T., Akerla, J., Honkanen, T., Krogerus, L., Salo, J., Paavonen, T., and Oksala, N. (2013) CIP2A expression and prognostic role in patients with esophageal adenocarcinoma. Med Oncol 30 132. Sung, W. W., Wang, Y. C., Lin, P. L., Cheng, Y. W., Chen, C. Y., Wu, T. C., and Lee, H. (2013) IL-10 Promotes Tumor Aggressiveness via Upregulation of CIP2A Transcription in Lung Adenocarcinoma. Clin Cancer Res 19, 4092-4103 133. Vaarala, M. H., Vaisanen, M. R., and Ristimaki, A. (2010) CIP2A expression is increased in prostate cancer. J Exp Clin Canc Res 29 134. Wiegering, A., Pfann, C., Uthe, F. W., Otto, C., Rycak, L., Mader, U., Gasser, M., Waaga-Gasser, A. M., Eilers, M., and Germer, C. T. (2013) CIP2A Influences Survival in Colon Cancer and Is Critical for Maintaining Myc Expression. Plos One 8 135. Yu, G. Z., Liu, G. H., Dong, J., and Jin, Y. Y. (2013) Clinical implications of CIP2A protein expression in breast cancer. Med Oncol 30 136. Zhai, M., Cong, L., Han, Y. X., and Tu, G. J. (2014) CIP2A is overexpressed in osteosarcoma and regulates cell proliferation and invasion. Tumor Biol 35, 1123- 1128 137. McCluskey, A., Sim, A. T., and Sakoff, J. A. (2002) Serine-threonine protein phosphatase inhibitors: development of potential therapeutic strategies. J Med Chem 45, 1151-1175 138. Swingle, M. R., Amable, L., Lawhorn, B. G., Buck, S. B., Burke, C. P., Ratti, P., Fischer, K. L., Boger, D. L., and Honkanen, R. E. (2009) Structure-activity relationship studies of fostriecin, cytostatin, and key analogs, with PP1, PP2A, PP5, and( beta12-beta13)-chimeras (PP1/PP2A and PP5/PP2A), provide further insight into the inhibitory actions of fostriecin family inhibitors. The Journal of pharmacology and experimental therapeutics 331, 45-53 139. Cho, U. S., Morrone, S., Sablina, A. A., Arroyo, J. D., Hahn, W. C., and Xu, W. (2007) Structural basis of PP2A inhibition by small t antigen. PLoS Biol 5, e202 140. Pallas, D. C., Shahrik, L. K., Martin, B. L., Jaspers, S., Miller, T. B., Brautigan, D. L., and Roberts, T. M. (1990) Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell 60, 167- 176 141. Kamibayashi, C., Estes, R., Lickteig, R. L., Yang, S. I., Craft, C., and Mumby, M. C. (1994) Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. J Biol Chem 269, 20139-20148 142. Beisiegel, U., Weber, W., Ihrke, G., Herz, J., and Stanley, K. K. (1989) The Ldl Receptor Related Protein, Lrp, Is an Apolipoprotein-E-Binding Protein. Nature 341, 162-164

121

143. Kowal, R. C., Herz, J., Goldstein, J. L., Esser, V., and Brown, M. S. (1989) Low- Density Lipoprotein Receptor-Related Protein Mediates Uptake of Cholesteryl Esters Derived from Apoprotein-E-Enriched Lipoproteins. Proc Natl Acad Sci U S A 86, 5810-5814 144. Brown, M. S., and Goldstein, J. L. (1986) A Receptor-Mediated Pathway for Cholesterol Homeostasis. Science 232, 34-47 145. Avila, E. M., Holdsworth, G., Sasaki, N., Jackson, R. L., and Harmony, J. A. K. (1982) Apoprotein-E Suppresses Phytohemagglutinin-Activated Phospholipid Turnover in Peripheral-Blood Mononuclear-Cells. Journal of Biological Chemistry 257, 5900-5909 146. Curtiss, L. K., and Edgington, T. S. (1981) The Biologic Activity of the Immunoregulatory Lipoprotein, Ldl-in, Is Independent of Its Free Fatty-Acid Content. J Immunol 126, 1382-1386 147. Pepe, M. G., and Curtiss, L. K. (1986) Apolipoprotein-E Is a Biologically-Active Constituent of the Normal Immunoregulatory Lipoprotein, Ldl-In. J Immunol 136, 3716-3723 148. Agarwal, A., MacKenzie, R. J., Pippa, R., Eide, C. A., Oddo, J., Tyner, J. W., Sears, R., Vitek, M. P., Odero, M. D., Christensen, D. J., and Druker, B. J. (2014) Antagonism of SET Using OP449 Enhances the Efficacy of Tyrosine Kinase Inhibitors and Overcomes Drug Resistance in Myeloid Leukemia. Clin Cancer Res 20, 2092-2103 149. Agarwal, A., MacKenzie, R., Oddo, J., Vitek, M. P., Christensen, D. J., and Druker, B. J. (2011) A Novel SET Antagonist (OP449) Is Cytotoxic to CML Cells, Including the Highly-Resistant BCR-ABL(T315I) Mutant, and Demonstrates Enhanced Efficacy in Combination with ABL Tyrosine Kinase Inhibitors. Blood 118, 1603-1604 150. Christensen, D. J., Ohkubo, N., Oddo, J., Van Kanegan, M. J., Neil, J., Li, F. Q., Colton, C. A., and Vitek, M. P. (2011) Apolipoprotein E and Peptide Mimetics Modulate Inflammation by Binding the SET Protein and Activating Protein Phosphatase 2A. J Immunol 186, 2535-2542 151. Switzer, C. H., Cheng, R. Y. S., Vitek, T. M., Christensen, D. J., Wink, D. A., and Vitek, M. P. (2011) Targeting SET/I(2)PP2A oncoprotein functions as a multi- pathway strategy for cancer therapy. Oncogene 30, 2504-2513 152. Suzuki, S., Li, X. K., Enosawa, S., and Shinomiya, T. (1996) A new immunosuppressant, FTY720, induces bcl-2-associated apoptotic cell death in human lymphocytes. Immunology 89, 518-523 153. Suzuki, S., Enosawa, S., Kakefuda, T., Shinomiya, T., Amari, M., Naoe, S., Hoshino, Y., and Chiba, K. (1996) A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation. Transplantation 61, 200-205 154. Brinkmann, V. (2009) FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects in the immune and the central nervous system. Brit J Pharmacol 158, 1173- 1182 155. Brinkmann, V., Davis, M. D., Heise, C. E., Albert, R., Cottens, S., Hof, R., Bruns, C., Prieschl, E., Baumruker, T., Hiestand, P., Foster, C. A., Zollinger, M., and

122

Lynch, K. R. (2002) The immune modulator FTY720 targets sphingosine 1- phosphate receptors. J Biol Chem 277, 21453-21457 156. Mandala, S., Hajdu, R., Bergstrom, J., Quackenbush, E., Xie, J., Milligan, J., Thornton, R., Shei, G. J., Card, D., Keohane, C., Rosenbach, M., Hale, J., Lynch, C. L., Rupprecht, K., Parsons, W., and Rosen, H. (2002) Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296, 346-349 157. Matsuoka, Y., Nagahara, Y., Ikekita, M., and Shinomiya, T. (2003) A novel immunosuppressive agent FTY720 induced Akt dephosphorylation in leukemia cells. Brit J Pharmacol 138, 1303-1312 158. Neviani, P., Santhanam, R., Oaks, J. J., Eiring, A. M., Notari, M., Blaser, B. W., Liu, S., Trotta, R., Muthusamy, N., Gambacorti-Passerini, C., Druker, B. J., Cortes, J., Marcucci, G., Chen, C. S., Verrills, N. M., Roy, D. C., Caligiuri, M. A., Bloomfield, C. D., Byrd, J. C., and Perrotti, D. (2007) FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphocytic leukemia. J Clin Invest 117, 2408-2421 159. Roberts, K. G., Smith, A. M., McDougall, F., Carpenter, H., Horan, M., Neviani, P., Powell, J. A., Thomas, D., Guthridge, M. A., Perrotti, D., Sim, A. T. R., Ashman, L. K., and Verrills, N. M. (2010) Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers. Cancer Res 70, 5438-5447 160. Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) Ceramide Activates Heterotrimeric Protein Phosphatase-2a. Journal of Biological Chemistry 268, 15523-15530 161. Hannun, Y. A., and Obeid, L. M. (2008) Principles of bioactive lipid signalling: lessons from sphingolipids. Nat Rev Mol Cell Bio 9, 139-150 162. Kim, S. W., Kim, H. J., Chun, Y. J., and Kim, M. Y. (2010) Ceramide Produces Apoptosis Through Induction of p27kip1 by Protein Phosphatase 2A-dependent Akt Dephosphorylation in PC-3 Prostate Cancer Cells. J Toxicol Env Heal A 73, 1465-1476 163. Lin, C. F., Chen, C. L., Chiang, C. W., Jan, M. S., Huang, W. C., and Lin, Y. S. (2007) GSK-3 beta acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 in ceramide-induced mitochondrial apoptosis. J Cell Sci 120, 2935-2943 164. Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Programmed Cell-Death Induced by Ceramide. Science 259, 1769-1771 165. Mukhopadhyay, A., Saddoughi, S. A., Song, P. F., Sultan, I., Ponnusamy, S., Senkal, C. E., Snook, C. F., Arnold, H. K., Sears, R. C., Hannun, Y. A., and Ogretmen, B. (2009) Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling. Faseb J 23, 751-763 166. Corson, T. W., and Crews, C. M. (2007) Molecular understanding and modern application of traditional medicines: Triumphs and trials. Cell 130, 769-774 167. Liu, Z., Ma, L., Wen, Z. S., Hu, Z., Wu, F. Q., Li, W., Liu, J. S., and Zhou, G. B. (2014) Cancerous inhibitor of PP2A is targeted by natural compound celastrol for degradation in non-small-cell lung cancer. Carcinogenesis 35, 905-914

123

168. Liu, Z., Ma, L., Wen, Z. S., Cheng, Y. X., and Zhou, G. B. (2014) Ethoxysanguinarine Induces Inhibitory Effects and Downregulates CIP2A in Lung Cancer Cells. Acs Med Chem Lett 5, 113-118 169. Li, W. G., Dai, F. Y., Cheng, Y. X., Yin, G. F., Bi, J. L., and Li, D. P. (2013) Identification of Porcine Reproductive and Respiratory Syndrome Virus Inhibitors Through an Oriented Screening on Natural Products. Chem Res Chinese U 29, 290- 293 170. Adams, J., Palombella, V. J., Sausville, E. A., Johnson, J., Destree, A., Lazarus, D. D., Maas, J., Pien, C. S., Prakash, S., and Elliott, P. J. (1999) Proteasome inhibitors: A novel class of potent and effective antitumor agents. Cancer Res 59, 2615-2622 171. Orlowski, R. Z., and Kuhn, D. J. (2008) Proteasome inhibitors in cancer therapy: Lessons from the first decade. Clin Cancer Res 14, 1649-1657 172. Tseng, L. M., Liu, C. Y., Chang, K. C., Chu, P. Y., Shiau, C. W., and Chen, K. F. (2012) CIP2A is a target of bortezomib in human triple negative breast cancer cells. Breast Cancer Res 14 173. Chen, K. F., Yeh, P. Y., Hsu, C., Hsu, C. H., Lu, Y. S., Hsieh, H. P., Chen, P. J., and Cheng, A. L. (2009) Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway. Journal of Biological Chemistry 284, 11121-11133 174. Chen, K. F., Pao, K. C., Su, J. C., Chou, Y. C., Liu, C. Y., Chen, H. J., Huang, J. W., Kim, I., and Shiau, C. W. (2012) Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity. Bioorgan Med Chem 20, 6144-6153 175. Arroyo, J. D., and Hahn, W. C. (2005) Involvement of PP2A in viral and cellular transformation. Oncogene 24, 7746-7755 176. Bryant, J. C., Westphal, R. S., and Wadzinski, B. E. (1999) Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory B alpha subunit. Biochem J 339, 241-246 177. Ogris, E., Gibson, D. M., and Pallas, D. C. (1997) Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. Oncogene 15, 911-917 178. Lee, J., Chen, Y., Tolstykh, T., and Stock, J. (1996) A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc Natl Acad Sci U S A 93, 6043-6047 179. Wei, H. J., Ashby, D. G., Moreno, C. S., Ogris, E., Yeong, F. M., Corbett, A. H., and Pallas, D. C. (2001) Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits CDC55p and RTS1p. Journal of Biological Chemistry 276, 1570-1577 180. Janssens, V., Goris, J., and Van Hoof, C. (2005) PP2A: the expected tumor suppressor. Curr Opin Genet Dev 15, 34-41 181. Lee, J., and Stock, J. (1993) Protein Phosphatase-2a Catalytic Subunit Is Methyl- Esterified at Its Carboxyl-Terminus by a Novel Methyltransferase. Journal of Biological Chemistry 268, 19192-19195 182. Guenin, S., Schwartz, L., Morvan, D., Steyaert, J. M., Poignet, A., Madelmont, J. C., and Demidem, A. (2008) PP2A activity is controlled by methylation and

124

regulates oncoprotein expression in melanoma cells: A mechanism which participates in growth inhibition induced by chloroethylnitrosourea treatment. Int J Oncol 32, 49-57 183. Godeneche, D., Rapp, M., Thierry, A., Laval, F., Madelmont, J. C., Chollet, P., and Veyre, A. (1990) DNA Damage Induced by a New 2-Chloroethyl Nitrosourea on Malignant-Melanoma Cells. Cancer Res 50, 5898-5903 184. Puustinen, P., Junttila, M. R., Vanhatupa, S., Sablina, A. A., Hector, M. E., Teittinen, K., Raheem, O., Ketola, K., Lin, S., Kast, J., Haapasalo, H., Hahn, W. C., and Westermarck, J. (2009) PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res 69, 2870-2877 185. Bachovchin, D. A., Mohr, J. T., Speers, A. E., Wang, C., Berlin, J. M., Spicer, T. P., Fernandez-Vega, V., Chase, P., Hodder, P. S., Schurer, S. C., Nomura, D. K., Rosen, H., Fu, G. C., and Cravatt, B. F. (2011) Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase- 1 inhibitors. Proc Natl Acad Sci U S A 108, 6811-6816 186. Bachovchin, D. A., Zuhl, A. M., Speers, A. E., Wolfe, M. R., Weerapana, E., Brown, S. J., Rosen, H., and Cravatt, B. F. (2011) Discovery and optimization of sulfonyl acrylonitriles as selective, covalent inhibitors of protein phosphatase methylesterase-1. J Med Chem 54, 5229-5236 187. Seamon, K. B., Padgett, W., and Daly, J. W. (1981) Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci U S A 78, 3363-3367 188. Cristobal, I., Garcia-Orti, L., Cirauqui, C., Alonso, M. M., Calasanz, M. J., and Odero, M. D. (2011) PP2A impaired activity is a common event in acute myeloid leukemia and its activation by forskolin has a potent anti-leukemic effect. Leukemia 25, 606-614 189. Cristobal, I., Rincon, R., Manso, R., Madoz-Gurpide, J., Carames, C., del Puerto- Nevado, L., Rojo, F., and Garcia-Foncillas, J. (2014) Hyperphosphorylation of PP2A in colorectal cancer and the potential therapeutic value showed by its forskolin-induced dephosphorylation and activation. Biochim Biophys Acta 1842, 1823-1829 190. Feschenko, M. S., Stevenson, E., Nairn, A. C., and Sweadner, K. J. (2002) A novel cAMP-stimulated pathway in protein phosphatase 2A activation. J Pharmacol Exp Ther 302, 111-118 191. Gutierrez, A., Pan, L., Groen, R. W., Baleydier, F., Kentsis, A., Marineau, J., Grebliunaite, R., Kozakewich, E., Reed, C., Pflumio, F., Poglio, S., Uzan, B., Clemons, P., VerPlank, L., An, F., Burbank, J., Norton, S., Tolliday, N., Steen, H., Weng, A. P., Yuan, H., Bradner, J. E., Mitsiades, C., Look, A. T., and Aster, J. C. (2014) Phenothiazines induce PP2A-mediated apoptosis in T cell acute lymphoblastic leukemia. J Clin Invest 124, 644-655 192. Kar, S., Palit, S., Ball, W. B., and Das, P. K. (2012) Carnosic acid modulates Akt/IKK/NF-kappa B signaling by PP2A and induces intrinsic and extrinsic pathway mediated apoptosis in human prostate carcinoma PC-3 cells. Apoptosis 17, 735-747

125

193. Birringer, M., EyTina, J. H., Salvatore, B. A., and Neuzil, J. (2003) Vitamin E analogues as inducers of apoptosis: structure-function relation. Br J Cancer 88, 1948-1955 194. Burton, G. W., and Traber, M. G. (1990) Vitamin-E - Antioxidant Activity, Biokinetics, and Bioavailability. Annu Rev Nutr 10, 357-382 195. Gu, X. B., Song, X. D., Dong, Y. H., Cai, H., Walters, E., Zhang, R. S., Pang, X. W., Xie, T. P., Guo, Y. H., Sridhar, R., and Califano, J. A. (2008) Vitamin E succinate induces ceramide-mediated apoptosis in head and neck squamous cell carcinoma in vitro and in vivo. Clin Cancer Res 14, 1840-1848 196. Lawson, K. A., Anderson, K., Menchaca, M., Atkinson, J., Sun, L. Z., Knight, V., Gilbert, B. E., Conti, C., Sanders, B. G., and Kline, K. (2003) Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis. Mol Cancer Ther 2, 437-444 197. Neuzil, J., Svensson, I., Weber, T., Weber, C., and Brunk, U. T. (1999) alpha- tocopheryl succinate-induced apoptosis in Jurkat T cells involves caspase-3 activation, and both lysosomal and mitochondrial destabilisation. FEBS Lett 445, 295-300 198. Neuzil, J., Tomasetti, M., Zhao, Y., Dong, L. F., Birringer, M., Wang, X. F., Low, P., Wu, K., Salvatore, B. A., and Ralph, S. J. (2007) Vitamin E analogs, a novel group of "mitocans," as anticancer agents: The importance of being redox-silent. Mol Pharmacol 71, 1185-1199 199. Qian, M., Kralova, J., Yu, W. P., Bose, H. R., Dvorak, M., Sanders, B. G., and Kline, K. (1997) c-Jun involvement in vitamin E succinate induced apoptosis of reticuloendotheliosis virus transformed avian lymphoid cells. Oncogene 15, 223- 230 200. Wang, X. F., Dong, L. F., Zhao, Y., Tomasetti, M., Wu, K., and Neuzil, J. (2006) Vitamin E analogues as anticancer agents: Lessons from studies with alpha- tocopheryl succinate. Mol Nutr Food Res 50, 675-685 201. You, H. H., Yu, W. P., Sanders, B. G., and Kline, K. (2001) RRR-alpha-tocopheryl succinate induces MDA-MB-435 and MCF-7 human breast cancer cells to undergo differentiation. Cell Growth Differ 12, 471-480 202. Yu, W. P., Liao, Q. Y., Hantash, F. M., Sanders, B. G., and Kline, K. (2001) Activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha- tocopheryl succinate-induced apoptosis of human breast cancer cells. Cancer Res 61, 6569-6576 203. Zhang, Y., Ni, J., Messing, E. M., Chang, E., Yang, C. R., and Yeh, S. Y. (2002) Vitamin E succinate inhibits the function of androgen receptor and the expression of prostate-specific antigen in prostate cancer cells. Proc Natl Acad Sci U S A 99, 7408-7413 204. Neuzil, J., Weber, T., Schroder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G. C., Olejnicka, B., Negre-Salvayre, A., Sticha, M., Coffey, R. J., and Weber, C. (2001) Induction of cancer cell apoptosis by alpha-tocopheryl succinate: molecular pathways and structural requirements. Faseb J 15, 403-415 205. Huang, P. H., Wang, D. S., Chuang, H. C., Wei, S., Kulp, S. K., and Chen, C. S. (2009) alpha-Tocopheryl succinate and derivatives mediate the transcriptional

126

repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK- Sp1-signaling axis. Carcinogenesis 30, 1125-1131 206. Sheiness, D., Fanshier, L., and Bishop, J. M. (1978) Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29. J Virol 28, 600-610 207. Roussel, M., Saule, S., Lagrou, C., Rommens, C., Beug, H., Graf, T., and Stehelin, D. (1979) Three new types of viral oncogene of cellular origin specific for haematopoietic cell transformation. Nature 281, 452-455 208. Alitalo, K., Ramsay, G., Bishop, J. M., Pfeifer, S. O., Colby, W. W., and Levinson, A. D. (1983) Identification of nuclear proteins encoded by viral and cellular myc oncogenes. Nature 306, 274-277 209. Vennstrom, B., Sheiness, D., Zabielski, J., and Bishop, J. M. (1982) Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29. J Virol 42, 773-779 210. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tronick, S., Aaronson, S., and Leder, P. (1982) Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc Natl Acad Sci U S A 79, 7837-7841 211. Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D., Gallo, R. C., and Croce, C. M. (1982) Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci U S A 79, 7824-7827 212. Land, H., Parada, L. F., and Weinberg, R. A. (1983) Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304, 596-602 213. Armelin, H. A., Armelin, M. C., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984) Functional role for c-myc in mitogenic response to platelet- derived growth factor. Nature 310, 655-660 214. Stone, J., de Lange, T., Ramsay, G., Jakobovits, E., Bishop, J. M., Varmus, H., and Lee, W. (1987) Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol 7, 1697-1709 215. Leone, G., DeGregori, J., Sears, R., Jakoi, L., and Nevins, J. R. (1997) Myc and Ras collaborate in inducing accumulation of active cyclin E/Cdk2 and E2F. Nature 387, 422-426 216. He, T. C., Sparks, A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Identification of c-MYC as a target of the APC pathway. Science 281, 1509-1512 217. Herbst, A., Hemann, M. T., Tworkowski, K. A., Salghetti, S. E., Lowe, S. W., and Tansey, W. P. (2005) A conserved element in Myc that negatively regulates its proapoptotic activity. EMBO Rep 6, 177-183 218. Arvanitis, C., and Felsher, D. W. (2006) Conditional transgenic models define how MYC initiates and maintains tumorigenesis. Seminars in cancer biology 16, 313- 317 219. Weng, A. P., Millholland, J. M., Yashiro-Ohtani, Y., Arcangeli, M. L., Lau, A., Wai, C., Del Bianco, C., Rodriguez, C. G., Sai, H., Tobias, J., Li, Y., Wolfe, M. S., Shachaf, C., Felsher, D., Blacklow, S. C., Pear, W. S., and Aster, J. C. (2006) c-

127

Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20, 2096-2109 220. Meyer, N., and Penn, L. Z. (2008) Reflecting on 25 years with MYC. Nat Rev Cancer 8, 976-990 221. Levens, D. (2010) You Don't Muck with MYC. Genes Cancer 1, 547-554 222. Dang, C. V. (2012) MYC on the path to cancer. Cell 149, 22-35 223. Gabay, M., Li, Y., and Felsher, D. W. (2014) MYC activation is a hallmark of cancer initiation and maintenance. Cold Spring Harb Perspect Med 4 224. Sears, R., Leone, G., DeGregori, J., and Nevins, J. R. (1999) Ras enhances Myc protein stability. Mol Cell 3, 169-179 225. Sears, R., Nuckolls, F., Haura, E., Taya, Y., Tamai, K., and Nevins, J. R. (2000) Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev 14, 2501-2514 226. D'Cruz, C. M., Gunther, E. J., Boxer, R. B., Hartman, J. L., Sintasath, L., Moody, S. E., Cox, J. D., Ha, S. I., Belka, G. K., Golant, A., Cardiff, R. D., and Chodosh, L. A. (2001) c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations. Nat Med 7, 235-239 227. Yu, Y., Dong, W., Li, X., Yu, E., Zhou, X., and Li, S. (2003) Significance of c- Myc and Bcl-2 protein expression in nasopharyngeal carcinoma. Arch Otolaryngol Head Neck Surg 129, 1322-1326 228. Palomero, T., Lim, W. K., Odom, D. T., Sulis, M. L., Real, P. J., Margolin, A., Barnes, K. C., O'Neil, J., Neuberg, D., Weng, A. P., Aster, J. C., Sigaux, F., Soulier, J., Look, A. T., Young, R. A., Califano, A., and Ferrando, A. A. (2006) NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci U S A 103, 18261-18266 229. DePinho, R., Mitsock, L., Hatton, K., Ferrier, P., Zimmerman, K., Legouy, E., Tesfaye, A., Collum, R., Yancopoulos, G., Nisen, P., and et al. (1987) Myc family of cellular oncogenes. J Cell Biochem 33, 257-266 230. DePinho, R. A., Schreiber-Agus, N., and Alt, F. W. (1991) myc family oncogenes in the development of normal and neoplastic cells. Adv Cancer Res 57, 1-46 231. Mukherjee, B., Morgenbesser, S. D., and DePinho, R. A. (1992) Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and trans-acting dominant mutants. Genes Dev 6, 1480-1492 232. Yeh, E., Cunningham, M., Arnold, H., Chasse, D., Monteith, T., Ivaldi, G., Hahn, W. C., Stukenberg, P. T., Shenolikar, S., Uchida, T., Counter, C. M., Nevins, J. R., Means, A. R., and Sears, R. (2004) A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nature cell biology 6, 308-318 233. Mongiardi, M. P., Savino, M., Falchetti, M. L., Illi, B., Bozzo, F., Valle, C., Helmer-Citterich, M., Ferre, F., Nasi, S., and Levi, A. (2016) c-MYC inhibition impairs hypoxia response in glioblastoma multiforme. Oncotarget 7, 33257-33271 234. Fiorentino, F. P., Tokgun, E., Sole-Sanchez, S., Giampaolo, S., Tokgun, O., Jauset, T., Kohno, T., Perucho, M., Soucek, L., and Yokota, J. (2016) Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation. Oncotarget 7, 31014-31028

128

235. Li, Y., Casey, S. C., and Felsher, D. W. (2014) Inactivation of MYC reverses tumorigenesis. J Intern Med 276, 52-60 236. Liu, J., Perumal, N. B., Oldfield, C. J., Su, E. W., Uversky, V. N., and Dunker, A. K. (2006) Intrinsic disorder in transcription factors. Biochemistry 45, 6873-6888 237. Tsafou, K., Tiwari, P. B., Forman-Kay, J. D., Metallo, S. J., and Toretsky, J. A. (2018) Targeting Intrinsically Disordered Transcription Factors: Changing the Paradigm. J Mol Biol 430, 2321-2341 238. Carabet, L. A., Rennie, P. S., and Cherkasov, A. (2018) Therapeutic Inhibition of Myc in Cancer. Structural Bases and Computer-Aided Drug Discovery Approaches. Int J Mol Sci 20 239. Wierstra, I., and Alves, J. (2008) The c-myc promoter: still MysterY and challenge. Adv Cancer Res 99, 113-333 240. Sears, R. C. (2004) The life cycle of C-myc: from synthesis to degradation. Cell cycle (Georgetown, Tex.) 3, 1133-1137 241. Arnold, H. K., and Sears, R. C. (2008) A tumor suppressor role for PP2A-B56alpha through negative regulation of c-Myc and other key oncoproteins. Cancer metastasis reviews 27, 147-158 242. Allen-Petersen, B. L., and Sears, R. C. (2019) Mission Possible: Advances in MYC Therapeutic Targeting in Cancer. BioDrugs 243. Zhang, X., Farrell, A. S., Daniel, C. J., Arnold, H., Scanlan, C., Laraway, B. J., Janghorban, M., Lum, L., Chen, D., Troxell, M., and Sears, R. (2012) Mechanistic insight into Myc stabilization in breast cancer involving aberrant Axin1 expression. Proc Natl Acad Sci U S A 109, 2790-2795 244. Farrell, A. S., Allen-Petersen, B., Daniel, C. J., Wang, X., Wang, Z., Rodriguez, S., Impey, S., Oddo, J., Vitek, M. P., Lopez, C., Christensen, D. J., Sheppard, B., and Sears, R. C. (2014) Targeting inhibitors of the tumor suppressor PP2A for the treatment of pancreatic cancer. Molecular cancer research : MCR 12, 924-939 245. Janghorban, M., Farrell, A. S., Allen-Petersen, B. L., Pelz, C., Daniel, C. J., Oddo, J., Langer, E. M., Christensen, D. J., and Sears, R. C. (2014) Targeting c-MYC by antagonizing PP2A inhibitors in breast cancer. Proc Natl Acad Sci U S A 111, 9157- 9162 246. Hemann, M. T., Bric, A., Teruya-Feldstein, J., Herbst, A., Nilsson, J. A., Cordon- Cardo, C., Cleveland, J. L., Tansey, W. P., and Lowe, S. W. (2005) Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436, 807-811 247. Wang, X., Cunningham, M., Zhang, X., Tokarz, S., Laraway, B., Troxell, M., and Sears, R. C. (2011) Phosphorylation regulates c-Myc's oncogenic activity in the mammary gland. Cancer Res 71, 925-936 248. Schleger, C., Verbeke, C., Hildenbrand, R., Zentgraf, H., and Bleyl, U. (2002) c- MYC activation in primary and metastatic ductal adenocarcinoma of the pancreas: incidence, mechanisms, and clinical significance. Mod Pathol 15, 462-469 249. Stathis, A., and Bertoni, F. (2018) BET Proteins as Targets for Anticancer Treatment. Cancer Discov 8, 24-36 250. Yang, Z., He, N., and Zhou, Q. (2008) Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol Cell Biol 28, 967-976

129

251. Ott, C. J., Kopp, N., Bird, L., Paranal, R. M., Qi, J., Bowman, T., Rodig, S. J., Kung, A. L., Bradner, J. E., and Weinstock, D. M. (2012) BET bromodomain inhibition targets both c-Myc and IL7R in high-risk acute lymphoblastic leukemia. Blood 120, 2843-2852 252. Grayson, A. R., Walsh, E. M., Cameron, M. J., Godec, J., Ashworth, T., Ambrose, J. M., Aserlind, A. B., Wang, H., Evan, G., Kluk, M. J., Bradner, J. E., Aster, J. C., and French, C. A. (2014) MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma. Oncogene 33, 1736-1742 253. Bandopadhayay, P., Bergthold, G., Nguyen, B., Schubert, S., Gholamin, S., Tang, Y., Bolin, S., Schumacher, S. E., Zeid, R., Masoud, S., Yu, F., Vue, N., Gibson, W. J., Paolella, B. R., Mitra, S. S., Cheshier, S. H., Qi, J., Liu, K. W., Wechsler-Reya, R., Weiss, W. A., Swartling, F. J., Kieran, M. W., Bradner, J. E., Beroukhim, R., and Cho, Y. J. (2014) BET bromodomain inhibition of MYC-amplified medulloblastoma. Clin Cancer Res 20, 912-925 254. Togel, L., Nightingale, R., Chueh, A. C., Jayachandran, A., Tran, H., Phesse, T., Wu, R., Sieber, O. M., Arango, D., Dhillon, A. S., Dawson, M. A., Diez-Dacal, B., Gahman, T. C., Filippakopoulos, P., Shiau, A. K., and Mariadason, J. M. (2016) Dual Targeting of Bromodomain and Extraterminal Domain Proteins, and WNT or MAPK Signaling, Inhibits c-MYC Expression and Proliferation of Colorectal Cancer Cells. Mol Cancer Ther 15, 1217-1226 255. Choi, S. K., Hong, S. H., Kim, H. S., Shin, C. Y., Nam, S. W., Choi, W. S., Han, J. W., and You, J. S. (2016) JQ1, an inhibitor of the epigenetic reader BRD4, suppresses the bidirectional MYC-AP4 axis via multiple mechanisms. Oncol Rep 35, 1186-1194 256. Delmore, J. E., Issa, G. C., Lemieux, M. E., Rahl, P. B., Shi, J., Jacobs, H. M., Kastritis, E., Gilpatrick, T., Paranal, R. M., Qi, J., Chesi, M., Schinzel, A. C., McKeown, M. R., Heffernan, T. P., Vakoc, C. R., Bergsagel, P. L., Ghobrial, I. M., Richardson, P. G., Young, R. A., Hahn, W. C., Anderson, K. C., Kung, A. L., Bradner, J. E., and Mitsiades, C. S. (2011) BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904-917 257. Zhang, Z., Ma, P., Jing, Y., Yan, Y., Cai, M. C., Zhang, M., Zhang, S., Peng, H., Ji, Z. L., Di, W., Gu, Z., Gao, W. Q., and Zhuang, G. (2016) BET Bromodomain Inhibition as a Therapeutic Strategy in Ovarian Cancer by Downregulating FoxM1. Theranostics 6, 219-230 258. Andrews, F. H., Singh, A. R., Joshi, S., Smith, C. A., Morales, G. A., Garlich, J. R., Durden, D. L., and Kutateladze, T. G. (2017) Dual-activity PI3K-BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis. Proc Natl Acad Sci U S A 114, E1072-E1080 259. Sun, K., Atoyan, R., Borek, M. A., Dellarocca, S., Samson, M. E., Ma, A. W., Xu, G. X., Patterson, T., Tuck, D. P., Viner, J. L., Fattaey, A., and Wang, J. (2017) Dual HDAC and PI3K Inhibitor CUDC-907 Downregulates MYC and Suppresses Growth of MYC-dependent Cancers. Mol Cancer Ther 16, 285-299 260. Gerlach, D., Tontsch-Grunt, U., Baum, A., Popow, J., Scharn, D., Hofmann, M. H., Engelhardt, H., Kaya, O., Beck, J., Schweifer, N., Gerstberger, T., Zuber, J., Savarese, F., and Kraut, N. (2018) The novel BET bromodomain inhibitor BI

130

894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML. Oncogene 37, 2687-2701 261. Tontsch-Grunt, U., Rudolph, D., Waizenegger, I., Baum, A., Gerlach, D., Engelhardt, H., Wurm, M., Savarese, F., Schweifer, N., and Kraut, N. (2018) Synergistic activity of BET inhibitor BI 894999 with PLK inhibitor volasertib in AML in vitro and in vivo. Cancer Lett 421, 112-120 262. Yang, D., and Hurley, L. H. (2006) Structure of the biologically relevant G- quadruplex in the c-MYC promoter. Nucleosides Nucleotides Nucleic Acids 25, 951-968 263. Hurley, L. H., Von Hoff, D. D., Siddiqui-Jain, A., and Yang, D. (2006) Drug targeting of the c-MYC promoter to repress gene expression via a G-quadruplex silencer element. Semin Oncol 33, 498-512 264. Brooks, T. A., and Hurley, L. H. (2010) Targeting MYC Expression through G- Quadruplexes. Genes Cancer 1, 641-649 265. Gonzalez, V., and Hurley, L. H. (2010) The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity. Biochemistry 49, 9706-9714 266. Mathad, R. I., Hatzakis, E., Dai, J., and Yang, D. (2011) c-MYC promoter G- quadruplex formed at the 5'-end of NHE III1 element: insights into biological relevance and parallel-stranded G-quadruplex stability. Nucleic Acids Res 39, 9023-9033 267. Hu, M. H., Wang, Y. Q., Yu, Z. Y., Hu, L. N., Ou, T. M., Chen, S. B., Huang, Z. S., and Tan, J. H. (2018) Discovery of a New Four-Leaf Clover-Like Ligand as a Potent c-MYC Transcription Inhibitor Specifically Targeting the Promoter G- Quadruplex. J Med Chem 61, 2447-2459 268. Shu, B., Cao, J., Kuang, G., Qiu, J., Zhang, M., Zhang, Y., Wang, M., Li, X., Kang, S., Ou, T. M., Tan, J. H., Huang, Z. S., and Li, D. (2018) Syntheses and evaluation of new acridone derivatives for selective binding of oncogene c-myc promoter i- motifs in gene transcriptional regulation. Chem Commun (Camb) 54, 2036-2039 269. Shu, B., Zeng, P., Kang, S., Li, P. H., Hu, D., Kuang, G., Cao, J., Li, X., Zhang, M., An, L. K., Huang, Z. S., and Li, D. (2019) Syntheses and evaluation of new Quinoline derivatives for inhibition of hnRNP K in regulating oncogene c-myc transcription. Bioorg Chem 85, 1-17 270. Bouvard, C., Lim, S. M., Ludka, J., Yazdani, N., Woods, A. K., Chatterjee, A. K., Schultz, P. G., and Zhu, S. (2017) Small molecule selectively suppresses MYC transcription in cancer cells. Proc Natl Acad Sci U S A 114, 3497-3502 271. Allen-Petersen, B. L., Risom, T., Feng, Z., Wang, Z., Jenny, Z. P., Thoma, M. C., Pelz, K. R., Morton, J. P., Sansom, O. J., Lopez, C. D., Sheppard, B., Christensen, D. J., Ohlmeyer, M., Narla, G., and Sears, R. C. (2019) Activation of PP2A and Inhibition of mTOR Synergistically Reduce MYC Signaling and Decrease Tumor Growth in Pancreatic Ductal Adenocarcinoma. Cancer Res 79, 209-219 272. Guo, J., Parise, R. A., Joseph, E., Egorin, M. J., Lazo, J. S., Prochownik, E. V., and Eiseman, J. L. (2009) Efficacy, pharmacokinetics, tisssue distribution, and metabolism of the Myc-Max disruptor, 10058-F4 [Z,E]-5-[4-ethylbenzylidine]-2- thioxothiazolidin-4-one, in mice. Cancer Chemother Pharmacol 63, 615-625

131

273. Wang, H., Chauhan, J., Hu, A., Pendleton, K., Yap, J. L., Sabato, P. E., Jones, J. W., Perri, M., Yu, J., Cione, E., Kane, M. A., Fletcher, S., and Prochownik, E. V. (2013) Disruption of Myc-Max heterodimerization with improved cell-penetrating analogs of the small molecule 10074-G5. Oncotarget 4, 936-947 274. Yap, J. L., Wang, H., Hu, A., Chauhan, J., Jung, K. Y., Gharavi, R. B., Prochownik, E. V., and Fletcher, S. (2013) Pharmacophore identification of c-Myc inhibitor 10074-G5. Bioorg Med Chem Lett 23, 370-374 275. Castell, A., Yan, Q., Fawkner, K., Hydbring, P., Zhang, F., Verschut, V., Franco, M., Zakaria, S. M., Bazzar, W., Goodwin, J., Zinzalla, G., and Larsson, L. G. (2018) A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation. Scientific reports 8, 10064 276. Struntz, N. B., Chen, A., Deutzmann, A., Wilson, R. M., Stefan, E., Evans, H. L., Ramirez, M. A., Liang, T., Caballero, F., Wildschut, M. H. E., Neel, D. V., Freeman, D. B., Pop, M. S., McConkey, M., Muller, S., Curtin, B. H., Tseng, H., Frombach, K. R., Butty, V. L., Levine, S. S., Feau, C., Elmiligy, S., Hong, J. A., Lewis, T. A., Vetere, A., Clemons, P. A., Malstrom, S. E., Ebert, B. L., Lin, C. Y., Felsher, D. W., and Koehler, A. N. (2019) Stabilization of the Max Homodimer with a Small Molecule Attenuates Myc-Driven Transcription. Cell Chem Biol 26, 711-723 e714 277. Soucek, L., Helmer-Citterich, M., Sacco, A., Jucker, R., Cesareni, G., and Nasi, S. (1998) Design and properties of a Myc derivative that efficiently homodimerizes. Oncogene 17, 2463-2472 278. Soucek, L., Jucker, R., Panacchia, L., Ricordy, R., Tato, F., and Nasi, S. (2002) Omomyc, a potential Myc dominant negative, enhances Myc-induced apoptosis. Cancer Res 62, 3507-3510 279. Savino, M., Annibali, D., Carucci, N., Favuzzi, E., Cole, M. D., Evan, G. I., Soucek, L., and Nasi, S. (2011) The action mechanism of the Myc inhibitor termed Omomyc may give clues on how to target Myc for cancer therapy. PLoS One 6, e22284 280. Annibali, D., Whitfield, J. R., Favuzzi, E., Jauset, T., Serrano, E., Cuartas, I., Redondo-Campos, S., Folch, G., Gonzalez-Junca, A., Sodir, N. M., Masso-Valles, D., Beaulieu, M. E., Swigart, L. B., Mc Gee, M. M., Somma, M. P., Nasi, S., Seoane, J., Evan, G. I., and Soucek, L. (2014) Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis. Nature communications 5, 4632 281. Whitfield, J. R., Beaulieu, M. E., and Soucek, L. (2017) Strategies to Inhibit Myc and Their Clinical Applicability. Frontiers in cell and developmental biology 5, 10 282. Wierstra, I., and Alves, J. (2008) Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1. Biochem Biophys Res Commun 368, 107-115 283. Takagi, Y., Futamura, M., Yamaguchi, K., Aoki, S., Takahashi, T., and Saji, S. (2000) Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers. Gut 47, 268-271 284. Esplin, E. D., Ramos, P., Martinez, B., Tomlinson, G. E., Mumby, M. C., and Evans, G. A. (2006) The glycine 90 to aspartate alteration in the Abeta subunit of PP2A (PPP2R1B) associates with breast cancer and causes a deficit in protein function. Genes Chromosomes Cancer 45, 182-190

132

285. Baysal, B. E., Willett-Brozick, J. E., Taschner, P. E., Dauwerse, J. G., Devilee, P., and Devlin, B. (2001) A high-resolution integrated map spanning the SDHD gene at 11q23: a 1.1-Mb BAC contig, a partial transcript map and 15 new repeat polymorphisms in a tumour-suppressor region. Eur J Hum Genet 9, 121-129 286. Bluemn, E. G., Spencer, E. S., Mecham, B., Gordon, R. R., Coleman, I., Lewinshtein, D., Mostaghel, E., Zhang, X., Annis, J., Grandori, C., Porter, C., and Nelson, P. S. (2013) PPP2R2C loss promotes castration-resistance and is associated with increased prostate cancer-specific mortality. Mol Cancer Res 11, 568-578 287. Fan, Y. L., Chen, L., Wang, J., Yao, Q., and Wan, J. Q. (2013) Over expression of PPP2R2C inhibits human glioma cells growth through the suppression of mTOR pathway. FEBS Lett 587, 3892-3897 288. Grochola, L. F., Vazquez, A., Bond, E. E., Wurl, P., Taubert, H., Muller, T. H., Levine, A. J., and Bond, G. L. (2009) Recent natural selection identifies a genetic variant in a regulatory subunit of protein phosphatase 2A that associates with altered cancer risk and survival. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 6301-6308 289. Beroukhim, R., Mermel, C. H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J. S., Dobson, J., Urashima, M., Mc Henry, K. T., Pinchback, R. M., Ligon, A. H., Cho, Y. J., Haery, L., Greulich, H., Reich, M., Winckler, W., Lawrence, M. S., Weir, B. A., Tanaka, K. E., Chiang, D. Y., Bass, A. J., Loo, A., Hoffman, C., Prensner, J., Liefeld, T., Gao, Q., Yecies, D., Signoretti, S., Maher, E., Kaye, F. J., Sasaki, H., Tepper, J. E., Fletcher, J. A., Tabernero, J., Baselga, J., Tsao, M. S., Demichelis, F., Rubin, M. A., Janne, P. A., Daly, M. J., Nucera, C., Levine, R. L., Ebert, B. L., Gabriel, S., Rustgi, A. K., Antonescu, C. R., Ladanyi, M., Letai, A., Garraway, L. A., Loda, M., Beer, D. G., True, L. D., Okamoto, A., Pomeroy, S. L., Singer, S., Golub, T. R., Lander, E. S., Getz, G., Sellers, W. R., and Meyerson, M. (2010) The landscape of somatic copy-number alteration across human cancers. Nature 463, 899-905 290. Ohshima, K., Hatakeyama, K., Nagashima, T., Watanabe, Y., Kanto, K., Doi, Y., Ide, T., Shimoda, Y., Tanabe, T., Ohnami, S., Ohnami, S., Serizawa, M., Maruyama, K., Akiyama, Y., Urakami, K., Kusuhara, M., Mochizuki, T., and Yamaguchi, K. (2017) Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors. Scientific reports 7, 641 291. Schaub, F. X., Dhankani, V., Berger, A. C., Trivedi, M., Richardson, A. B., Shaw, R., Zhao, W., Zhang, X., Ventura, A., Liu, Y., Ayer, D. E., Hurlin, P. J., Cherniack, A. D., Eisenman, R. N., Bernard, B., and Grandori, C. (2018) Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas. Cell systems 6, 282-300.e282 292. Adhikary, S., and Eilers, M. (2005) Transcriptional regulation and transformation by Myc proteins. Nature reviews. Molecular cell biology 6, 635-645 293. Chen, H., Liu, H., and Qing, G. (2018) Targeting oncogenic Myc as a strategy for cancer treatment. Signal transduction and targeted therapy 3, 5 294. Soucek, L., Nasi, S., and Evan, G. I. (2004) Omomyc expression in skin prevents Myc-induced papillomatosis. Cell Death Differ 11, 1038-1045

133

295. Soucek, L., Whitfield, J. R., Sodir, N. M., Masso-Valles, D., Serrano, E., Karnezis, A. N., Swigart, L. B., and Evan, G. I. (2013) Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice. Genes Dev 27, 504-513 296. Jung, L. A., Gebhardt, A., Koelmel, W., Ade, C. P., Walz, S., Kuper, J., von Eyss, B., Letschert, S., Redel, C., d'Artista, L., Biankin, A., Zender, L., Sauer, M., Wolf, E., Evan, G., Kisker, C., and Eilers, M. (2017) OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors. Oncogene 36, 1911-1924 297. Arroyo, J. D., and Hahn, W. C. (2005) Involvement of PP2A in viral and cellular transformation. Oncogene 24, 7746-7755 298. Chen, W., Arroyo, J. D., Timmons, J. C., Possemato, R., and Hahn, W. C. (2005) Cancer-associated PP2A Aalpha subunits induce functional haploinsufficiency and tumorigenicity. Cancer Res 65, 8183-8192 299. Sablina, A. A., and Hahn, W. C. (2007) The role of PP2A A subunits in tumor suppression. Cell adhesion & migration 1, 140-141 300. Janssens, V., and Goris, J. (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. The Biochemical journal 353, 417-439 301. Janssens, V., Goris, J., and Van Hoof, C. (2005) PP2A: the expected tumor suppressor. Current opinion in genetics & development 15, 34-41 302. Lambrecht, C., Libbrecht, L., Sagaert, X., Pauwels, P., Hoorne, Y., Crowther, J., Louis, J. V., Sents, W., Sablina, A., and Janssens, V. (2018) Loss of protein phosphatase 2A regulatory subunit B56delta promotes spontaneous tumorigenesis in vivo. Oncogene 37, 544-552 303. Sents, W., Meeusen, B., Kalev, P., Radaelli, E., Sagaert, X., Miermans, E., Haesen, D., Lambrecht, C., Dewerchin, M., Carmeliet, P., Westermarck, J., Sablina, A., and Janssens, V. (2017) PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development. Cancer Res 77, 6825-6837 304. Arnold, H. K., and Sears, R. C. (2006) Protein phosphatase 2A regulatory subunit B56alpha associates with c-myc and negatively regulates c-myc accumulation. Mol Cell Biol 26, 2832-2844 305. Escamilla-Powers, J. R., and Sears, R. C. (2007) A conserved pathway that controls c-Myc protein stability through opposing phosphorylation events occurs in yeast. J Biol Chem 282, 5432-5442 306. Agarwal, A., MacKenzie, R. J., Pippa, R., Eide, C. A., Oddo, J., Tyner, J. W., Sears, R., Vitek, M. P., Odero, M. D., Christensen, D. J., and Druker, B. J. (2014) Antagonism of SET using OP449 enhances the efficacy of tyrosine kinase inhibitors and overcomes drug resistance in myeloid leukemia. Clin Cancer Res 20, 2092-2103 307. Arriazu, E., Pippa, R., and Odero, M. D. (2016) Protein Phosphatase 2A as a Therapeutic Target in Acute Myeloid Leukemia. Frontiers in oncology 6, 78 308. Hu, X., Garcia, C., Fazli, L., Gleave, M., Vitek, M. P., Jansen, M., Christensen, D., and Mulholland, D. J. (2015) Inhibition of Pten deficient Castration Resistant Prostate Cancer by Targeting of the SET - PP2A Signaling axis. Scientific reports 5, 15182

134

309. Neviani, P., and Perrotti, D. (2014) SETting OP449 into the PP2A-activating drug family. Clin Cancer Res 20, 2026-2028 310. O'Connor, C. M., Perl, A., Leonard, D., Sangodkar, J., and Narla, G. (2018) Therapeutic targeting of PP2A. The international journal of biochemistry & cell biology 96, 182-193 311. Pippa, R., Dominguez, A., Christensen, D. J., Moreno-Miralles, I., Blanco-Prieto, M. J., Vitek, M. P., and Odero, M. D. (2014) Effect of FTY720 on the SET-PP2A complex in acute myeloid leukemia; SET binding drugs have antagonistic activity. Leukemia 28, 1915-1918 312. Ramaswamy, K., Spitzer, B., and Kentsis, A. (2015) Therapeutic Re-Activation of Protein Phosphatase 2A in Acute Myeloid Leukemia. Frontiers in oncology 5, 16 313. Richard, N. P., Pippa, R., Cleary, M. M., Puri, A., Tibbitts, D., Mahmood, S., Christensen, D. J., Jeng, S., McWeeney, S., Look, A. T., Chang, B. H., Tyner, J. W., Vitek, M. P., Odero, M. D., Sears, R., and Agarwal, A. (2016) Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia. Oncotarget 7, 84214-84227 314. Sangodkar, J., Farrington, C. C., McClinch, K., Galsky, M. D., Kastrinsky, D. B., and Narla, G. (2016) All roads lead to PP2A: exploiting the therapeutic potential of this phosphatase. The FEBS journal 283, 1004-1024 315. Kastrinsky, D. B., Sangodkar, J., Zaware, N., Izadmehr, S., Dhawan, N. S., Narla, G., and Ohlmeyer, M. (2015) Reengineered tricyclic anti-cancer agents. Bioorganic & medicinal chemistry 23, 6528-6534 316. Kauko, O., O'Connor, C. M., Kulesskiy, E., Sangodkar, J., Aakula, A., Izadmehr, S., Yetukuri, L., Yadav, B., Padzik, A., Laajala, T. D., Haapaniemi, P., Momeny, M., Varila, T., Ohlmeyer, M., Aittokallio, T., Wennerberg, K., Narla, G., and Westermarck, J. (2018) PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells. Science translational medicine 10 317. McClinch, K., Avelar, R. A., Callejas, D., Izadmehr, S., Wiredja, D., Perl, A., Sangodkar, J., Kastrinsky, D. B., Schlatzer, D., Cooper, M., Kiselar, J., Stachnik, A., Yao, S., Hoon, D., McQuaid, D., Zaware, N., Gong, Y., Brautigan, D. L., Plymate, S. R., Sprenger, C. C. T., Oh, W. K., Levine, A. C., Kirschenbaum, A., Sfakianos, J. P., Sears, R., DiFeo, A., Ioannou, Y., Ohlmeyer, M., Narla, G., and Galsky, M. D. (2018) Small-Molecule Activators of Protein Phosphatase 2A for the Treatment of Castration-Resistant Prostate Cancer. Cancer Res 78, 2065-2080 318. Sangodkar, J., Perl, A., Tohme, R., Kiselar, J., Kastrinsky, D. B., Zaware, N., Izadmehr, S., Mazhar, S., Wiredja, D. D., O'Connor, C. M., Hoon, D., Dhawan, N. S., Schlatzer, D., Yao, S., Leonard, D., Borczuk, A. C., Gokulrangan, G., Wang, L., Svenson, E., Farrington, C. C., Yuan, E., Avelar, R. A., Stachnik, A., Smith, B., Gidwani, V., Giannini, H. M., McQuaid, D., McClinch, K., Wang, Z., Levine, A. C., Sears, R. C., Chen, E. Y., Duan, Q., Datt, M., Haider, S., Ma'ayan, A., DiFeo, A., Sharma, N., Galsky, M. D., Brautigan, D. L., Ioannou, Y. A., Xu, W., Chance, M. R., Ohlmeyer, M., and Narla, G. (2017) Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth. J Clin Invest 127, 2081-2090 319. Tohme, R., Izadmehr, S., Gandhe, S., Tabaro, G., Vallabhaneni, S., Thomas, A., Vasireddi, N., Dhawan, N. S., Ma'ayan, A., Sharma, N., Galsky, M. D., Ohlmeyer,

135

M., Sangodkar, J., and Narla, G. (2019) Direct activation of PP2A for the treatment of tyrosine kinase inhibitor-resistant lung adenocarcinoma. JCI Insight 4 320. Hummel, M., Bentink, S., Berger, H., Klapper, W., Wessendorf, S., Barth, T. F., Bernd, H. W., Cogliatti, S. B., Dierlamm, J., Feller, A. C., Hansmann, M. L., Haralambieva, E., Harder, L., Hasenclever, D., Kuhn, M., Lenze, D., Lichter, P., Martin-Subero, J. I., Moller, P., Muller-Hermelink, H. K., Ott, G., Parwaresch, R. M., Pott, C., Rosenwald, A., Rosolowski, M., Schwaenen, C., Sturzenhofecker, B., Szczepanowski, M., Trautmann, H., Wacker, H. H., Spang, R., Loeffler, M., Trumper, L., Stein, H., Siebert, R., and Molecular Mechanisms in Malignant Lymphomas Network Project of the Deutsche, K. (2006) A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling. N Engl J Med 354, 2419-2430 321. Johnson, L., Mercer, K., Greenbaum, D., Bronson, R. T., Crowley, D., Tuveson, D. A., and Jacks, T. (2001) Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 410, 1111-1116 322. Farrell, A. S., Joly, M. M., Allen-Petersen, B. L., Worth, P. J., Lanciault, C., Sauer, D., Link, J., Pelz, C., Heiser, L. M., Morton, J. P., Muthalagu, N., Hoffman, M. T., Manning, S. L., Pratt, E. D., Kendsersky, N. D., Egbukichi, N., Amery, T. S., Thoma, M. C., Jenny, Z. P., Rhim, A. D., Murphy, D. J., Sansom, O. J., Crawford, H. C., Sheppard, B. C., and Sears, R. C. (2017) MYC regulates ductal- neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance. Nature communications 8, 1728 323. Walz, S., Lorenzin, F., Morton, J., Wiese, K. E., von Eyss, B., Herold, S., Rycak, L., Dumay-Odelot, H., Karim, S., Bartkuhn, M., Roels, F., Wustefeld, T., Fischer, M., Teichmann, M., Zender, L., Wei, C. L., Sansom, O., Wolf, E., and Eilers, M. (2014) Activation and repression by oncogenic MYC shape tumour-specific gene expression profiles. Nature 511, 483-487 324. Richardson, G. E., and Johnson, B. E. (1993) The biology of lung cancer. Semin Oncol 20, 105-127 325. Wolfer, A., Wittner, B. S., Irimia, D., Flavin, R. J., Lupien, M., Gunawardane, R. N., Meyer, C. A., Lightcap, E. S., Tamayo, P., Mesirov, J. P., Liu, X. S., Shioda, T., Toner, M., Loda, M., Brown, M., Brugge, J. S., and Ramaswamy, S. (2010) MYC regulation of a "poor-prognosis" metastatic cancer cell state. Proc Natl Acad Sci U S A 107, 3698-3703 326. Seo, A. N., Yang, J. M., Kim, H., Jheon, S., Kim, K., Lee, C. T., Jin, Y., Yun, S., Chung, J. H., and Paik, J. H. (2014) Clinicopathologic and prognostic significance of c-MYC copy number gain in lung adenocarcinomas. Br J Cancer 110, 2688- 2699 327. Fukazawa, T., Maeda, Y., Matsuoka, J., Yamatsuji, T., Shigemitsu, K., Morita, I., Faiola, F., Durbin, M. L., Soucek, L., and Naomoto, Y. (2010) Inhibition of Myc effectively targets KRAS mutation-positive lung cancer expressing high levels of Myc. Anticancer research 30, 4193-4200 328. Fallah, Y., Brundage, J., Allegakoen, P., and Shajahan-Haq, A. N. (2017) MYC- Driven Pathways in Breast Cancer Subtypes. Biomolecules 7

136

329. Yang, A., Qin, S., Schulte, B. A., Ethier, S. P., Tew, K. D., and Wang, G. Y. (2017) MYC Inhibition Depletes Cancer Stem-like Cells in Triple-Negative Breast Cancer. Cancer Res 77, 6641-6650 330. Wang, E., Sorolla, A., Cunningham, P. T., Bogdawa, H. M., Beck, S., Golden, E., Dewhurst, R. E., Florez, L., Cruickshank, M. N., Hoffmann, K., Hopkins, R. M., Kim, J., Woo, A. J., Watt, P. M., and Blancafort, P. (2018) Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers. Oncogene 331. Horiuchi, D., Kusdra, L., Huskey, N. E., Chandriani, S., Lenburg, M. E., Gonzalez- Angulo, A. M., Creasman, K. J., Bazarov, A. V., Smyth, J. W., Davis, S. E., Yaswen, P., Mills, G. B., Esserman, L. J., and Goga, A. (2012) MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition. J Exp Med 209, 679-696 332. Lustig, L. C., Dingar, D., Tu, W. B., Lourenco, C., Kalkat, M., Inamoto, I., Ponzielli, R., Chan, W. C. W., Shin, J. A., and Penn, L. Z. (2017) Inhibiting MYC binding to the E-box DNA motif by ME47 decreases tumour xenograft growth. Oncogene 36, 6830-6837 333. Lee, J., Lim, B., Pearson, T., Choi, K., Fuson, J. A., Bartholomeusz, C., Paradiso, L. J., Myers, T., Tripathy, D., and Ueno, N. T. (2019) Anti-tumor and anti- metastasis efficacy of E6201, a MEK1 inhibitor, in preclinical models of triple- negative breast cancer. Breast Cancer Res Treat 175, 339-351 334. Nagaria, T. S., Shi, C., Leduc, C., Hoskin, V., Sikdar, S., Sangrar, W., and Greer, P. A. (2017) Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems. Oncotarget 8, 80804- 80819 335. Giovannelli, P., Di Donato, M., Galasso, G., Di Zazzo, E., Bilancio, A., and Migliaccio, A. (2018) The Androgen Receptor in Breast Cancer. Frontiers in endocrinology 9, 492 336. Barfeld, S. J., Urbanucci, A., Itkonen, H. M., Fazli, L., Hicks, J. L., Thiede, B., Rennie, P. S., Yegnasubramanian, S., DeMarzo, A. M., and Mills, I. G. (2017) c- Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks. EBioMedicine 18, 83-93 337. Sun, X. X., Sears, R. C., and Dai, M. S. (2015) Deubiquitinating c-Myc: USP36 steps up in the nucleolus. Cell cycle (Georgetown, Tex.) 14, 3786-3793 338. Fernandez, P. C., Frank, S. R., Wang, L., Schroeder, M., Liu, S., Greene, J., Cocito, A., and Amati, B. (2003) Genomic targets of the human c-Myc protein. Genes Dev 17, 1115-1129 339. Li, Z., Van Calcar, S., Qu, C., Cavenee, W. K., Zhang, M. Q., and Ren, B. (2003) A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells. Proc Natl Acad Sci U S A 100, 8164-8169 340. Qu, J., Zhao, X., Wang, J., Liu, X., Yan, Y., Liu, L., Cai, H., Qu, H., Lu, N., Sun, Y., Wang, F., Wang, J., and Zhang, J. (2017) MYC overexpression with its prognostic and clinicopathological significance in breast cancer. Oncotarget 8, 93998-94008 341. Tansey, W. P. (2014) Mammalian MYC Proteins and Cancer. New Journal of Science 2014, 27

137

342. Beltran, H. (2014) The N-myc Oncogene: Maximizing its Targets, Regulation, and Therapeutic Potential. Molecular Cancer Research 12, 7 343. Yue, Z. X., Huang, C., Gao, C., Xing, T. Y., Liu, S. G., Li, X. J., Zhao, Q., Wang, X. S., Zhao, W., Jin, M., and Ma, X. L. (2017) MYCN amplification predicts poor prognosis based on interphase fluorescence in situ hybridization analysis of bone marrow cells in bone marrow metastases of neuroblastoma. Cancer Cell Int 17, 43 344. Lee, W. H., Murphree, A. L., and Benedict, W. F. (1984) Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 309, 458-460 345. MacPherson, D., Conkrite, K., Tam, M., Mukai, S., Mu, D., and Jacks, T. (2007) Murine bilateral retinoblastoma exhibiting rapid-onset, metastatic progression and N-myc gene amplification. The EMBO journal 26, 784-794 346. Pfister, S., Remke, M., Benner, A., Mendrzyk, F., Toedt, G., Felsberg, J., Wittmann, A., Devens, F., Gerber, N. U., Joos, S., Kulozik, A., Reifenberger, G., Rutkowski, S., Wiestler, O. D., Radlwimmer, B., Scheurlen, W., Lichter, P., and Korshunov, A. (2009) Outcome prediction in pediatric medulloblastoma based on DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci. J Clin Oncol 27, 1627-1636 347. Swartling, F. J., Grimmer, M. R., Hackett, C. S., Northcott, P. A., Fan, Q. W., Goldenberg, D. D., Lau, J., Masic, S., Nguyen, K., Yakovenko, S., Zhe, X. N., Gilmer, H. C., Collins, R., Nagaoka, M., Phillips, J. J., Jenkins, R. B., Tihan, T., Vandenberg, S. R., James, C. D., Tanaka, K., Taylor, M. D., Weiss, W. A., and Chesler, L. (2010) Pleiotropic role for MYCN in medulloblastoma. Genes Dev 24, 1059-1072 348. Beltran, H., Rickman, D. S., Park, K., Chae, S. S., Sboner, A., MacDonald, T. Y., Wang, Y., Sheikh, K. L., Terry, S., Tagawa, S. T., Dhir, R., Nelson, J. B., de la Taille, A., Allory, Y., Gerstein, M. B., Perner, S., Pienta, K. J., Chinnaiyan, A. M., Wang, Y., Collins, C. C., Gleave, M. E., Demichelis, F., Nanus, D. M., and Rubin, M. A. (2011) Molecular characterization of neuroendocrine prostate cancer and identification of new drug targets. Cancer Discov 1, 487-495 349. Mosquera, J. M., Beltran, H., Park, K., MacDonald, T. Y., Robinson, B. D., Tagawa, S. T., Perner, S., Bismar, T. A., Erbersdobler, A., Dhir, R., Nelson, J. B., Nanus, D. M., and Rubin, M. A. (2013) Concurrent AURKA and MYCN gene amplifications are harbingers of lethal treatment-related neuroendocrine prostate cancer. Neoplasia 15, 1-10 350. Nau, M. M., Brooks, B. J., Jr., Carney, D. N., Gazdar, A. F., Battey, J. F., Sausville, E. A., and Minna, J. D. (1986) Human small-cell lung cancers show amplification and expression of the N-myc gene. Proc Natl Acad Sci U S A 83, 1092-1096 351. Wong, A. J., Ruppert, J. M., Eggleston, J., Hamilton, S. R., Baylin, S. B., and Vogelstein, B. (1986) Gene amplification of c-myc and N-myc in small cell carcinoma of the lung. Science 233, 461-464 352. Rickman, D. S., Schulte, J. H., and Eilers, M. (2018) The Expanding World of N- MYC-Driven Tumors. Cancer Discov 8, 150-163 353. Buchel, G., Carstensen, A., Mak, K. Y., Roeschert, I., Leen, E., Sumara, O., Hofstetter, J., Herold, S., Kalb, J., Baluapuri, A., Poon, E., Kwok, C., Chesler, L., Maric, H. M., Rickman, D. S., Wolf, E., Bayliss, R., Walz, S., and Eilers, M. (2017) Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and

138

Pause Release of RNA Polymerase II during the Cell Cycle. Cell reports 21, 3483- 3497 354. Richards, M. W., Burgess, S. G., Poon, E., Carstensen, A., Eilers, M., Chesler, L., and Bayliss, R. (2016) Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors. Proc Natl Acad Sci U S A 113, 13726-13731 355. Xiao, D., Yue, M., Su, H., Ren, P., Jiang, J., Li, F., Hu, Y., Du, H., Liu, H., and Qing, G. (2016) Polo-like Kinase-1 Regulates Myc Stabilization and Activates a Feedforward Circuit Promoting Tumor Cell Survival. Mol Cell 64, 493-506 356. Minna, J. D., Battey, J. F., Brooks, B. J., Cuttitta, F., Gazdar, A. F., Johnson, B. E., Ihde, D. C., Lebacq-Verheyden, A. M., Mulshine, J., Nau, M. M., and et al. (1986) Molecular genetic analysis reveals chromosomal deletion, gene amplification, and autocrine growth factor production in the pathogenesis of human lung cancer. Cold Spring Harb Symp Quant Biol 51 Pt 2, 843-853 357. Wang, J., Okkeri, J., Pavic, K., Wang, Z., Kauko, O., Halonen, T., Sarek, G., Ojala, P. M., Rao, Z., Xu, W., and Westermarck, J. (2017) Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56. EMBO Rep 18, 437- 450 358. Soofiyani, S. R., Hejazi, M. S., and Baradaran, B. (2017) The role of CIP2A in cancer: A review and update. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 96, 626-633 359. Wang, H., Choe, M. H., Lee, I. W., Namgoong, S., Kim, J. S., Kim, N. H., and Oh, J. S. (2017) CIP2A acts as a scaffold for CEP192-mediated microtubule organizing center assembly by recruiting Plk1 and aurora A during meiotic maturation. Development 144, 3829-3839 360. Wolfer, A., and Ramaswamy, S. (2010) Prognostic signatures, cancer metastasis and MYC. Cell cycle (Georgetown, Tex.) 9, 3639 361. Blando, J., Moore, T., Hursting, S., Jiang, G., Saha, A., Beltran, L., Shen, J., Repass, J., Strom, S., and DiGiovanni, J. (2011) Dietary energy balance modulates prostate cancer progression in Hi-Myc mice. Cancer Prev Res (Phila) 4, 2002-2014 362. Dejure, F. R., and Eilers, M. (2017) MYC and tumor metabolism: chicken and egg. The EMBO journal 36, 3409-3420 363. Wolpaw, A. J., and Dang, C. V. (2018) MYC-induced metabolic stress and tumorigenesis. Biochimica et biophysica acta. Reviews on cancer 1870, 43-50 364. Kauko, O., and Westermarck, J. (2018) Non-genomic mechanisms of protein phosphatase 2A (PP2A) regulation in cancer. The international journal of biochemistry & cell biology 96, 157-164 365. Holohan, C., Van Schaeybroeck, S., Longley, D. B., and Johnston, P. G. (2013) Cancer drug resistance: an evolving paradigm. Nat Rev Cancer 13, 714-726 366. Lu, T., Jackson, M. W., Singhi, A. D., Kandel, E. S., Yang, M., Zhang, Y., Gudkov, A. V., and Stark, G. R. (2009) Validation-based insertional mutagenesis identifies lysine demethylase FBXL11 as a negative regulator of NFkappaB. Proc Natl Acad Sci U S A 106, 16339-16344

139