Activity of the Second Generation BTK Inhibitor Acalabrutinib in Canine and
Human B-cell Non-Hodgkin Lymphoma
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Bonnie Kate Harrington
Graduate Program in Comparative and Veterinary Medicine
The Ohio State University
2018
Dissertation Committee
John C. Byrd, M.D., Advisor
Amy J. Johnson, Ph.D.
Krista La Perle, D.V.M., Ph.D.
William C. Kisseberth, D.V.M., Ph.D.
1
Copyrighted by
Bonnie Kate Harrington
2018
2
Abstract
Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton’s
Tyrosine Kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In these studies, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy reported to be similar to human
diffuse large B-cell lymphoma (DLBCL), as well as primary human chronic
lymphocytic leukemia (CLL) cells. We demonstrated that acalabrutinib potently inhibited BTK activity and downstream B-cell receptor (BCR) effectors in CLBL1, a canine B-cell lymphoma cell line, primary canine lymphoma cells, and primary
CLL cells. Compared to ibrutinib, acalabrutinib is a more specific inhibitor and lacked off-target effects on the T-cell and NK cell kinase IL-2 inducible T-cell kinase (ITK) and epidermal growth factor receptor (EGFR). Accordingly, acalabrutinib did not antagonize antibody dependent cell cytotoxicity mediated by
NK cells. Finally, acalabrutinib inhibited proliferation and viability in CLBL1 cells and primary CLL cells and abrogated chemotactic migration in primary CLL cells.
To support our in vitro findings, we conducted a clinical trial using companion dogs with spontaneously occurring B-cell lymphoma. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with
ii
adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL).
iii
Dedication
This work is dedicated to my father, Jeff Harrington, a survivor of both CLL and renal carcinoma; to my mother, Sandy Harrington; to my husband, Alan
Flechtner; and to my son, Blaise Flechtner. Thank you for supporting me.
iv
Acknowledgments
I would like to acknowledge my mentors Dr. John Byrd and Amy Johnson for providing the scholarly yet nurturing environment for my intellect to grow; the funding sources that provided my stipend during training, the National Institutes of Health Oncology T32 and the Pelotonia Graduate Fellowship; and all of those in the Experimental Hematology Lab or elsewhere that supported me in graduate school and contributed to my development as an independent investigator.
v
Vita
December 27, 1984……………………………………….Born – North Canton, Ohio
2003……………..……………………..…………………………GlenOak High School
2007………………………………….…B.S. Psychology, The Ohio State University
2012……………………………..…………………D.V.M., The Ohio State University
2012-Present…………..………………Ph.D. Candidate, The Ohio State University
PUBLICATIONS
Ozer H.G., El-Gamal D., Ben Powell B., Hing Z.A., Blachly J.S., Harrington
B.K., Mitchell S., Grieselhuber N.R.,Williams K., Lai T., Alinari L., Baiocchi R.A.,
Brinton L., Baskin E., Cannon M., Beaver L., Goettl, V.M., Lucas D.M., Woyach
J.A., Sampath D., Lehman A.M., Yu L.,Zhang J., Ma Y., Zhang Y., Spevak W.,
Shi S., Severson P., Shellooe R., Carias H., Tsang G., Dong K., Ewing T.,
Marimuthu A., Tantoy C., Walters L., Sanftner L., Rezaei H., Nespi M., Matusow
B., Habets G., Ibrahim P., Zhang C., Mathé1 E.A., Bollag G., Byrd J.C.,
Lapalombella R.(2018) BRD4 profiling identifies critical chronic lymphocytic leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor. Cancer Discov. 4, 458-477.
vi
Tsai Y.T., Lakshmanan A., Lehman A., Harrington B.K., McClanahan Lucas F.,
Tran M., Sass E.J., Long M., Flechtner A.D., Jaynes F., La Perle K., Coppola V.,
Lozanski G., Muthusamy N., Byrd J.C., Grever M., Lucas D.M. (2017)
BRAFV600E accelerates disease progression and enhances immune
suppression in a mouse model of B-cell leukemia. Blood Adv. 24, 2147-2160.
Chen T.L., Tran M., Lakshmanan A., Harrington B.K., Goettl V.M., Lehman A.M.,
Trudeau S., Lucas D.M., Johnson A.J., Byrd J.C., Hertlein E. (2017). NF-κB p50
(nfkb1) contributes to pathogenesis in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. Blood 130, 376-379.
Herman S.E.M., Montraveta A., Niemann C.U., Mora-Jensen H., Gulrajani M.,
Krantz F., Mantel R., Smith L.L., McClanahan F., Harrington B.K., Colomer D.,
Covey T., Byrd J.C., Izumi R., Kaptein A., Ulrich R., Johnson A.J., Lannutti B.J.,
Wiestner A., Woyach J.A. (2017). The Bruton Tyrosine Kinase (BTK) Inhibitor
Acalabrutinib Demonstrates Potent On-Target Effects and Efficacy in Two Mouse
Models of Chronic Lymphocytic Leukemia. Clin. Cancer Res. 23, 2831-284.
Harrington, B.K., Gardner, H.L., Izumi, R., Hamdy, A., Rothbaum, W., Coombes,
K., Kaptein, A., Covery, T., Gulrajani, M., Van Lith, B., Krejsa, C., Russell, D.,
Zhang, X., Urie, B., London,C.A., Byrd, J.C., Johnson, A.J., Kisseberth, W.C.
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(2016). ACP-196: A second generation BTK inhibitor in a canine model of B-cell non-Hodgkin lymphoma. PLoS One. 11, 7.
Byrd, J.C.*, Harrington, B.*, O’Brien, S., Jones, J.A., Schuh, A., Devereux, S.,
Chaves, J., Wierda, W.G., Awan, F.T., Brown, J.R., et al. (2016). Acalabrutinib
(ACP-196) in Relapsed Chronic Lymphocytic Leukemia. N. Engl. J. Med. 374,
323–332.
* Indicates these authors contributed equally
Stiff A., Trikha P., Wesolowski R., Kendra K., Hsu V., Uppati S., McMichael E.,
Duggan M, Campbell A, Keller K., Landi I., Zhong Y., Dubovsky J., Howard J.H.,
Yu L, Harrington B., Old M., Reiff S., Mace T., Tridandapani S., Muthusamy N.,
Caligiuri M.A., Byrd J.C., Carson W.E. 3rd. (2016). Myeloid-Derived Suppressor
Cells Express Bruton's Tyrosine Kinase and Can Be Depleted in Tumor-Bearing
Hosts by Ibrutinib Treatment. Cancer Res. 76, 2125-2136.
Coble, D.J., Shoemaker, M., Harrington, B., Dardenne, A.D., and Bolon, B.
(2015). Histiocytic Sarcoma and Bilateral Facial Vein Thrombosis in a Siberian
Hamster (Phodopus sungorus). Comp. Med. 65, 127–132.
Dubovsky, J.A., Flynn, R., Du, J., Harrington, B.K., Zhong, Y., Kaffenberger, B.,
Yang, C., Towns, W.H., Lehman, A., Johnson, A.J., et al. (2014). Ibrutinib
viii
treatment ameliorates murine chronic graft-versus-host disease. J. Clin. Invest.
124, 4867–4876.
Woyach, J.A., Bojnik, E., Ruppert, A.S., Stefanovski, M.R., Goettl, V.M.,
Smucker, K.A., Smith, L.L., Dubovsky, J.A., Towns, W.H., MacMurray, J.,
Harrington, B.K. et al. (2014). Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia
(CLL). Blood 123, 1207–1213.
Fenger, J.M., Bear, M.D., Volinia, S., Lin, T.-Y., Harrington, B.K., London, C.A., and Kisseberth, W.C. (2014). Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis. BMC Cancer 14,
84.
Zhong, Y., El-Gamal, D., Dubovsky, J.A., Beckwith, K.A., Harrington, B.K.,
Williams, K.E., Goettl, V.M., Jha, S., Mo, X., Jones, J.A., et al. (2014). Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia 28, 1158–1163.
Dubovsky, J.A., Chappell, D.L., Harrington, B.K., Agrawal, K., Andritsos, L.A.,
Flynn, J.M., Jones, J.A., Paulaitis, M.E., Bolon, B., Johnson, A.J., et al. (2013).
ix
Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane- associated antigen critical to niche homing. Blood 122, 3308–3316.
Fields of Study
Major Field: Comparative and Veterinary Medicine
x
Table of Contents
Abstract ...... ii Dedication ...... iv Acknowledgments ...... v Vita ...... vi Table of Contents ...... xi List of Tables...... xiii List of Figures ...... xiv Chapter 1. Introduction...... 1 1.1 Chronic Lymphocytic Leukemia ...... 1 1.2 Traditional Therapies for Lymphoid Malignancies ...... 3 1.3 Targeting Bruton’s Tyrosine Kinase (BTK) in B-cell Malignancies ...... 4 1.4 Exploiting Canine Lymphoma as a Model for Human Disease...... 6 Chapter 2. Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib (ACP- 196) in Canine Models of B-cell Non-Hodgkin Lymphoma ...... 12 2.1 Introduction ...... 12 2.2 Materials and Methods ...... 14 2.3 Results ...... 23 2.4 Discussion...... 28 2.5 Figures and Tables ...... 34 Chapter 3. Preclinical Evaluation of Acalabrutinib (ACP-196) in Chronic Lymphocytic Leukemia...... 46 3.1 Introduction...... 46 3.2 Materials and Methods ...... 47 3.3 Results ...... 50 3.4 Discussion...... 54 3.5 Figures and Tables ...... 56 xi
Chapter 4. Conclusions and Future Directions ...... 61 4.1 Circumventing BTK Inhibitor Resistance in CLL...... 61 4.2 Understanding and Circumventing BTK Inhibitor Resistance in Aggressive Lymphoma...... 64 Bibliography ...... 71
xii
List of Tables
Table 1. Patient demographics...... 41
Table 2. Percentage BTK target occupancy in peripheral blood B-cells and lymphoma aspirates of dogs following treatment with acalabrutinib...... 42
Table 3. Adverse events listed by grade and frequency...... 43
Table 4. Clinical response rates...... 44
xiii
List of Figures
Figure 1. Effects of acalabrutinib inhibitors on canine lymphoma cells...... 34
Figure 2. CLBL1 clusters with ABC-like canine lymphoma subtype...... 36
Figure 3. Histopathology of peripheral lymph node biopsies from dogs enrolled in
the acalabrutinib clinical trial...... 37
Figure 4. Pharmacokinetic data...... 38
Figure 5. Reduced target lesion size in acalabrutinib treated dogs...... 39
Figure 6. Progression free survival...... 40
Figure 7. Acalabrutinib (ACP-196) is a specific inhibitor of BTK...... 56
Figure 8. Chemokine dependent migration of primary CLL cells treated with
acalabrutinib...... 58
Figure 9. Acalabrutinib (ACP-196) induces modest apoptosis in CLL cells...... 59
Figure 10. Acalabrutinib ( ACP-196) does not inhibit NK cell mediated ADCC. ... 60
xiv
Chapter 1. Introduction
1.1 Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia (CLL) represents the most prevalent adult leukemia in western countries and is characterized by accumulation of a clonal population of mature B-cells in the peripheral blood and solid tissues. Though the clinical course is often indolent with many patients surviving 10 years or more,
CLL is considered incurable outside of allogeneic stem cell transplant, a therapy reserved for only young, fit patients, and all patients will eventually succumb to their cancer. Current statistical surveys suggest there are greater than 20,000 new cases each year in the U.S. and that 4,500 of these result in death.1 It is a
disease predominantly of the elderly, with the median age of 72 at diagnosis, and
it occurs more commonly in males than females.2 According to the guidelines put
forth by the International Workshop on CLL, diagnosis is established using blood
counts and differentials, a blood smear, and immunophenotyping analysis. The presence of ≥5,000 neoplastic B-cells/uL of blood for a duration not less than 3 months represents the principle criterion for diagnosis, though involvement of
solid tissues such as lymph nodes, spleen, bone marrow, and liver is not
uncommon.3 Immunophenotypic markers that distinguish CLL from other B-cell
malignancies include CD19, CD20, CD23, as well as the T-cell antigen CD5; low
1
levels of IgM, IgD, and CD79 may also be present.4 Cytology of the peripheral blood is also important for diagnosis and most often reveals numerous small lymphocytes with condensed chromatin and a thin border of cytoplasm, as well as “smudge cells” which are frail, broken cells caused by reduced levels of the structural protein vimentin.5 Histologically, the lymph node is infiltrated by
numerous small lymphocytes often forming proliferation centers, called
“pseudofollicles”.4 Chemotactic migration of malignant cells to pseudofollicles is
mediated by interaction of CXCR4 and CXCR5 on the surface of CLL cells with
their respective ligands, CXCL12 and CXCL13.6
Like many cancers, CLL is heterogenous with respect to disease
progression and prognosis. In effort to stratify patients into risk groups and guide
therapeutic intervention, prognostic staging systems have been developed, the most commonly applies ones being Rai and Binet.7,8 Both systems consider lymph node enlargement, organ infiltration, and cytopenias to predict patient
outcome and guide the initiation of treatment. The introduction of new prognostic factors has led to refinement of this as part of the CLL International
Prognostication Index. This system incorporates four additional variables on top of the existing clinical staging system to better predict patient outcome and, in particular, time to first treatment. These variables include age, TP53 status, IGHV mutational status, and serum β2-microglobulin.9
As many cases of CLL follow an indolent clinical course, immediate
treatment is often unnecessary nor has it been shown to improve patient
2
outcomes.3,10 Thus, these asymptomatic patients are monitored until clinical
disease progression necessitates initiation of therapy.
1.2 Traditional Therapies for Lymphoid Malignancies
Current first line treatment for CLL is tailored to the individual patient and
includes combinations of chemotherapeutic and immunotherapeutic agents.11
Commonly used chemotherapeutics include nucleoside analogues, such as fludarabine, pentostatin, and cladribine; other cytotoxic agents, such as cyclophosphamide, bendamustine and vincristine; steroids, such as prednisone; and the anthracycline doxorubicin. In recent years, monoclonal antibodies targeting CD20 (rituximab, ofatumumab, obinutuzumab) and CD52
(alemtuzumab) have also been included in some treatment regimens. First line therapy for young CLL patients exhibiting symptomatic disease typically involves the combination of fludarabine, cyclophosphamide, and rituximab (FCR).12 In spite of their efficacy, FCR regimens often lead to persistent cytopenias and profound immunosuppression, and resulting recurrent infections leading to death in some patients. In older patients, fludarabine regimens are not well tolerated, and bendamustine may substituted as an appropriate alkylating agent.13
While chemotherapeutics remain the mainstay of many cancer treatment regimens, they are often administered in the face of significant side effects, leading to decreased quality of life, and therapies are limited for relapsed patients.14 Additionally, survey studies suggest these therapies have resulted in
3
only modestly improved survival in some subsets of patients, particularly those who are young and/or demonstrate more advanced disease.11,15 As such, the need for improved treatments has long been recognized and has resulted in the
development of targeted therapeutics.
1.3 Targeting Bruton Agammaglobulinemia Tyrosine Kinase (BTK) in B-cell
Malignancies
Unlike chronic myeloid leukemia that is characterized by a common BCR-
ABL translocation that has been effectively targeted by the small molecules
inhibitor imatinib with great impact on survival, NHL and CLL lack a ubiquitous
genetic target. However, B-cell receptor (BCR) signaling and its importance to
tumor cell survival in NHL and CLL have been well-documented.16–24 Immediately down-stream of the BCR is the kinase BTK, which is essential for activation of several survival pathways including AKT25, ERK26, and NFκB27,28. In addition,
BTK has been shown to be essential to TLR9 signaling via MYD8829 and chemokine mediated homing and adhesion of B-cells in the microenvironment30.
All of these signaling pathways are activated in and contribute to the progression
of CLL and NHL (reviewed in 31). Mutation of the BTK gene in humans causes
the inherited disease X-linked agammaglobulinemia with absent peripheral B-
cells and severe immune deficiency, further providing evidence that BTK is an
integral component of the pathway.32,33 Given the importance of BCR signaling in
NHL and CLL and central role of BTK in this pathway, targeting this kinase represents an attractive strategy. 4
Development of selective small molecule inhibitors of BTK was initially challenging. The first BTK inhibitor to enter the clinic was the orally bioavailable, irreversibly binding drug ibrutinib.34 In vitro, ibrutinib disrupts signaling through
BTK resulting in reduced activity of downstream targets, including PI3K, NFκB, and MAPK, and reduced cellular activation and proliferation.34–36 A phase I study of ibrutinib in B-cell malignancies was initiated where high (>95%) target occupancy and durable clinical activity were noted in NHL and 9 of 16 CLL/SLL
(small lymphocytic leukemia) patients.37 No dose limiting toxicity was identified and overall toxicity was modest. Ibrutinib is now approved for CLL,
Waldenstrom’s macroglobulinemia, mantle cell lymphoma (MCL), follicular lymphoma, and graft vs. host disease. It has also shown activity in diffuse large
B-cell lymphoma (DLBCL)38, and clinical trials are ongoing. In all of these
indications, significant activity was observed and prompted breakthrough drug
designation by the FDA.
Though toxicities are generally reduced in comparison with standard
chemoimmunotherapy, ibrutinib administration occasionally results in unfavorable
side effects, many of which are attributable to its effects on off-target kinases.39,40
Off-target inhibition of IL-2 inducible T-cell kinase (ITK) is associated with
impaired NK cell and T-cell response, both of which are essential for antitumor
immunity39. In addition, ibrutinib inhibits epidermal growth factor receptor
(EGFR)40, which may contribute to the rash and diarrhea associated with this
treatment. Finally, inhibition of the kinase TEC in platelets leads to reduced
5
platelet activation and prolonged clotting, resulting in clinically relevant petechial or massive hemorrhage in some patients.41
To this effect, acalabrutinib was pursued for clinical development. A
second generation BTK inhibitor, acalabrutinib binds irreversibly to BTK with
comparable potency to ibrutinib.42 However, acalabrutinib avoids many of the
adverse events observed with ibrutinib given its improved target selectivity.41
Acalabrutinib lacks ITK and EGFR as alternative targets and has more favorable
metabolism and absent CYP3A/4A interactions compared to ibrutinib.41 Greater
target selectivity coupled with comparable potency to other drugs in its class
suggest this second generation BTK inhibitor has potential to improve the clinical
results observed with ibrutinib while diminishing the toxicity observed.
Acalabrutinib is now approved in relapsed/refractory MCL and is in clinical trials
for other B-cell malignancies.
1.4 Exploiting Canine Lymphoma as a Model for Human Disease
Non-Hodgkin lymphoma (NHL) is one of the most commonly occurring
malignancies in the dog. With the development and advancement of molecular
techniques, distinct subcategories of NHL are increasingly recognized. The
World Health Organization published its first consensus on NHL classifications
for humans in 20014, and subsequent immunophenotypic and genetic studies in both humans and dogs allowed adaptation of the human WHO classification to the canine in 201143. It is now well-recognized that in the Western world, diffuse
6
large B-cell lymphoma (DLBCL) is the most common NHL subtype occurring in dogs.
Homologies between the two species are numerous. Clinically, humans and canines commonly present with involvement of multiple lymph nodes and in advanced stages, bone marrow, spleen, liver and other solid tissues.44,45 In dogs,
the disease follows an aggressive clinical course, and without therapy essentially
all dogs succumb to death. Chemotherapy-based regimens, typically “CHOP”
(cyclophosphamide, doxorubicin, vincristine, and prednisone), are the mainstay
therapy for both species, though in humans, monoclonal antibodies are often
included in the regimen. Histologic characteristics are also similar.4,44 Perhaps
most importantly, emerging data support a similar cellular biology and molecular
pathogenesis. For example, exome sequencing of a small group of dogs
revealed somatic mutations in a number of known cancer drivers that are
affected in human DLBCL, such as MYC, POT1, and TRAF3, among others.46 In
2000, human DLBCL was separated into two prognostic categories, activated B-
cell (ABC) and germinal center B-cell (GCB), based on gene expression profiling.47 In these investigations, ABC patients exhibited significantly shorter
survival times and an attenuated response to anthracycline-based chemotherapy
regimens in comparison with GCB patients. These findings may be related to the
presence of upregulated pro-proliferation and pro-survival pathways, including
NfκB and BCR signaling, in the ABC category.48 In the dog, gene expression analysis has shown activation of similar prosurvival pathways like NF-κB, and
7
has indicated that most cases of canine DLBCL are similar to the ABC subtype, which carries important information regarding prognosis and treatment strategies.49–51
In addition to these obvious similarities, the canine offers many
advantages as a disease model. Broadly, the range of disease processes, from
cancers to endocrinopathies to genetic diseases, is similar between dogs and humans. In many cases, not the least of which is DLBCL, correlations have been drawn between human diseases and their counterparts in dogs. Since dogs develop these diseases spontaneously, they better replicate the molecular
complexity and disease heterogeneity seen in the human population in comparison to rodent models. These diseases arise in many dogs in absence of an inbred syngeneic background and in the presence of an intact immune system, ideal for studying tumor microenvironment. Shared living space between pet dogs and their owners also enables the study the role of environmental factors in disease, making them exemplary models for identifying the role of environmental exposures. Exposure to household chemicals52–54, fumes from
waste incinerators55, radioactive materials56, and magnetic fields57 have all been
implicated in both canine and human cancer, and in many cases, canines have
served as the sentinel animals for identifying the hazardous agents.
The requisite technical tools and databases to support molecular
applications are increasingly being developed and available for application in the dog. The canine genome sequence is available in full, and SNP databases are
8
emerging, such as Dog Genome SNP Database and Dog SNPs published by the
Broad Institute.58,59 Major biomedical suppliers have expanded their reagent
inventory to include numerous canine specific products, including antibodies,
PCR primers, exome capture arrays, microarray chips, and the list continues.
Demand from pet owners seeking medical care for their beloved pets has
initiated the development and optimization of many new clinical laboratory assays and diagnostic techniques for this species, so disease progression and response to therapy can be easily monitored in a clinical trial or toxicology study.
In comparison with rodent models, large body and size and the amenable temperament of dogs make them ideal for collection of large tissues samples and/or serial tissue sampling.
Irrespective of the primary endpoint of the study, there are numerous examples in which pre-clinical evaluation of a drug in the dog has resulted in successful advancement of the compound as a human therapeutic.34,41,60–62
Establishment of the Comparative Oncology Trials Consortium (COTC), a
component of the National Cancer Institute’s Comparative Oncology Program
(NCI-COP), in 2004 represents a landmark in the advancement of the canine
model for cancer drug development.63 The consortium unites pharmaceutical
development companies with over 20 academic institutions across the United
States with the mission to “design and execute clinical trials in dogs with cancer to assess novel therapies”.63 Infrastructure provided from the NCI enables execution of multi-institutional studies and ensures robust trial design with ample
9
collection of pharmacokinetic and pharmacodynamic data, and data acquired
through this mechanism directly inform subsequent human clinical trials. The
COTC is, thus, unifying human and veterinary oncology practice in a way never
before seen under the common goal of advancing knowledge of cancer biology
and promoting oncology therapeutic development.
Amidst the numerous advantages offered by a canine model, one must
also consider the drawbacks. In comparison with the traditional rodent models, dog are larger and, thus, require more drug and in the case of toxicology studies a considerably larger per diem to cover the cost of housing and food. Prolonged lifespan may necessitate longer treatment times, also translating to higher cost,
and slows the acquisition of data. When performing clinical trials in companion
dogs, assessment of therapeutic efficacy using survival data is complicated by
owner elected euthanasia and owner elected trial withdrawal for reasons
unrelated to disease progression. While the similarities between human and
canine physiology vastly outweigh the differences, one must consider key
biological differences. Differences in absorption, metabolism and excretion may
hinder translational value of pharmacokinetic assessments, and in the current era
of targeted therapeutics, protein/gene homology and species cross reactivity are
important to consider.
Overall, the benefits of using this canine model in many instances
outweigh these disadvantages. For the above reasons, we elected to use a
10
canine model to evaluate safety and efficacy of the novel therapeutic acalabrutinib.
11
Chapter 2. Preclinical Evaluation of the Novel BTK Inhibitor Acalabrutinib (ACP- 196) in Canine Models of B-cell Non-Hodgkin Lymphoma
2.1 Introduction
BCR signaling is a critical factor in the progression of many subtypes of B- cell NHL. This signaling is driven through a variety of mechanisms, including
BCR binding to self or foreign antigen64–70, overexpression or aberrant expression of signal transducers16,71, and oncogenic somatic mutations driving
distal signaling pathways72,73. Regardless of the mechanism of activation,
signaling via the BCR and the key proximal signaling molecule BTK leads to
increased cell proliferation, survival, and homing to the microenvironment.74–76
Several targeted therapeutics that inhibit this signaling pathway are in development, including those that target BTK. The clinical activity of
IMBRUVICA® (ibrutinib), a first-in-class BTK inhibitor, has validated BTK as a
therapeutic target in B-cell malignancies.
Second-generation BTK inhibitors with more selective kinase activity
profiles are being developed, including acalabrutinib (Acerta Pharma BV, Oss,
the Netherlands). Acalabrutinib covalently binds BTK at the cysteine-481 residue
and inhibits with greater in vivo potency and selectivity than ibrutinib77 and also has demonstrated efficacy in early clinical trials involving relapsed and refractory
CLL.78 Preclinical development of ibrutinib included treatment of dogs with B-cell
lymphoma34, perhaps because many similarities to human NHL are recapitulated
in canine B-cell lymphoma, including histologic characteristics and response to
12
chemotherapeutics. The life expectancy of untreated dogs with aggressive
disease is ~6 weeks.79 In humans, DLBCL is the most common subtype of NHL,
and the advent of genomic technologies has allowed molecular subtyping of this
heterogeneous disease process in both people and dogs.49–51 Gene expression
profiling (GEP) of canine DLBCL demonstrates that it can be genetically
subcategorized, similar to its human counterpart51, and that canine DLBCL is most similar to the ABC-like subgroup, often expressing an activated NFκB pathway50. Similar to DLBCL in humans, differences in progression-free and
overall survival were found between the ABC-like and GCB-like canine patients.
For these reasons, we elected to use a canine model of B-cell NHL to
evaluate the pharmacodynamic effects of acalabrutinib in vitro and in vivo. In this
study, we explore BCR signaling activity in canine neoplastic B-cells, and
demonstrate that acalabrutinib inhibits BTK signaling in vitro, resulting in
cytotoxic and anti-proliferative effects similar to those reported with ibrutinib.35
We compare the GEP of the canine lymphoma cell line CLBL1, with GEP from primary canine B-cell lymphomas, to establish ABC-like or GCB-like subtype in the in vitro model. Finally, we report results of a phase I clinical trial of acalabrutinib in canine B-cell lymphoma, showing clinical benefit in a significant
proportion of patients. Overall, our data demonstrate that acalabrutinib is a
clinically well-tolerated and effective BTK inhibitor in dogs.
13
2.2 Materials and Methods
Reagents
Acalabrutinib was provided by Acerta Pharma (Oss, Netherlands).
Fluorescein isothiocyanate (FITC)-labeled annexin-V and propidium iodide (PI)
for flow cytometry were purchased from BD Pharmingen (San Diego, CA).
Patient sample processing and cell culture
Biopsies and fine needle aspirates (FNAs) were collected from affected
peripheral lymph nodes of canine lymphoma patients. The canine B-cell
lymphoma line CLBL1 was a generous gift from Barbara Rütgen (Vienna,
Austria).80 Erythrocyte contamination was removed from FNAs by ammonium
chloride lysis. Peripheral blood samples were processed by density gradient
centrifugation, followed by positive magnetic enrichment of B-cells using anti-
canine CD21-PE antibody (clone CA2.1D6, Bio-Rad, Hercules, CA), and anti- phycoerythrin microbeads (Miltenyi Biotec, San Diego, CA). Enrichment was confirmed by flow cytometry. All samples were snap-frozen and stored in liquid nitrogen. Cell lines and primary lymphoma cells were incubated at 37°C with 5%
CO2 in RPMI-1640 medium enriched with 10% fetal bovine serum (Sigma-
Aldrich, St. Louis, MO), 100 U/mL penicillin/100 μg/mL streptomycin (Sigma-
Aldrich), and 2 mM L-glutamate (Invitrogen, Carlsbad, CA). For in vitro signaling,
apoptosis and proliferation experiments, cells were incubated with acalabrutinib
for 1 hour followed by 2 washes with phosphate buffered saline (PBS). For 120
14
hour experiments, cells were treated every 24 hours, washed, and returned to
the culture plate.
Immunoblot analysis
Cell lines and primary cells were treated with acalabrutinib and stimulated
with plate-bound anti-human IgM (MP Biomedicals; Santa Ana, CA) in order to
activate BCR signaling. Plates were prepared by incubating a 10 µg/mL IgM
solution in PBS for 6 to 12 hours at 4̊ C, and then rinsing with PBS. Cells in complete media were applied to the plate, centrifuged at 400 RPM for 4 minutes at room temperature, and incubated at 37 degrees C for 11 minutes. Whole cell lysates were then prepared as previously described 81, followed by polyacrylamide gel electrophoresis and transfer of proteins to nitrocellulose membranes. The following polyclonal antibodies were used to detect protein on immunoblots: anti-phospho-PLCG2 (Tyr 1217, Cat. #3871), anti-PLCG2 (Cat.
#3872), anti-phospho-IKBA (Ser32, Cat. #2859), anti-IKBA (Cat. #4812), anti- phospho-ERK1/2 (Thr202/Tyr204, Cat. #9101), anti-ERK1/2 (Cat. #9102), anti- phospho-AKT (Thr308, Cat. #9257), and anti-AKT (Cat. #9272), anti-phospho-
NFKB P65 (Ser536, Cat. #3031), anti-NFKB P65 (Cat. #3034)(Cell Signaling
Technologies; Danvers, MA), anti-phospho-BTK (Tyr223, Cat. #ab68217,
Abcam, Cambridge, MA), and anti-BTK (cat. #B3187, Sigma-Aldrich).
15
Viability and proliferation assays
Cell viability was measured using annexin-V/PI flow cytometry (Beckman-
Coulter; Miami, FL). Cell proliferation was measured using Click-iT® Plus EdU
Alexa Fluor® 647 Flow Cytometry Assay Kit (Life Technologies, Grand Island,
NY) according to manufacturer instructions. Staining and analysis were performed as previously described by our laboratory.17
RNA extraction and gene expression profiling
Total RNA was isolated using the Trizol method and DNase treated. RNA integrity was interrogated using the Agilent 2100 Bioanalyzer (Agilent
Technologies, Palo Alto, CA). A 2 µg aliquot of total RNA was linearly amplified and labeled using the BioArray High Yield RNA Transcript labeling kit (Enzo Life
Sciences). Then, 15 µg of labeled cRNA was fragmented following the manufacturer instructions. Labeled cRNA targets were hybridized to Affymetrix
GeneChip® Canine Genome 2.0 array for 16 hours at 45 °C rotating at 60 rpm.
Arrays were washed and stained using the Fluidics Station 450 and scanned using the GeneChip Scanner 3000 (Affymetrix). For gene expression analysis, arrays were normalized. The microarray data have been deposited in the NCBI
Gene Expression Omnibus (GSE81110).
For clustering, we downloaded supplementary microarray data
(GSE43664) from GEO, then merged the microarray data for CLBL1 and
16
processed the combined data using the robust multiarray averaging (RMA)
method in version 3.1.2 of the R statistical programming environment. We then
applied the median polish algorithm to the subset of 1180 canine probe sets
found to separate two subtypes of canine B-cell lymphoma.50 Samples were clustered using centroid linkage and a distance metric based on un-centered correlation. To confirm the cluster assignment, we computed distance from
CLBL1 to the centers of both the GCB and ABC clusters using three methods:
Euclidean, Pearson correlation, and un-centered correlation.
Study Design
This clinical trial was a multicenter open-label, nonrandomized, sequential group, dose-escalation study of acalabrutinib in companion dogs with spontaneous B-cell lymphoma. The study was approved by The Ohio State
University Veterinary Medical Center Clinical Research Advisory Committee and
Institutional Animal Care and Use Committee. Similar permission was obtained at Pittsburgh Veterinary Specialty and Emergency Center. Owners gave written informed consent prior to patient enrollment. Treatment-naïve dogs and dogs with prior therapies and a confirmed diagnosis of new or relapsed B-cell lymphoma (stage ≥ 2) were eligible. Diagnosis of B-cell lymphoma was based on lymph node fine needle aspirate (FNA) cytology and/or histologic evaluation of lymph node biopsies and flow cytometry, immunophenotyping, immunohistochemistry, or PCR for antigen receptor rearrangement.82 Dogs met
17
all eligibility criteria, including: ≥1 year of age; performance status of 0-1;
adequate organ function as determined by routine bloodwork; ≥2 weeks from
previous anti-neoplastic treatments (chemotherapy, radiation therapy, surgery, or other investigational therapies) and complete resolution of toxicities from prior treatments. Dogs with T-cell lymphoma were excluded. Prior to enrollment,
dogs underwent screening tests including complete blood count, serum
biochemistry profile, urinalysis, thoracic radiographs, tumor immunophenotyping
and lymph node biopsy for evaluation of histomorphologic subtype.
Dogs with spontaneous B-cell lymphoma that failed conventional therapy or for which there was no therapeutic alternative, or for which conventional therapy was not desired by the owner were enrolled in the study. Dogs were administered acalabrutinib orally (PO) once or twice daily for a 7 day cycle and continued treatment until clinical progression. Assessment of patients for tumor
response was performed weekly by direct tumor measurement, or through the
use of imaging techniques such as radiography or ultrasound. Assessment of
clinical toxicities and tumor response were performed at each visit. Dogs were
evaluated for hematologic and biochemical toxicities every 7 days with routine
bloodwork (CBC, serum biochemical profile). The initial dose of 2.5 mg/kg orally
once daily was based on a previous data from a toxicology study in healthy
purpose bred dogs (data not shown) and dose escalations were set at 5, 10, 15,
and 20 mg/kg once or twice daily in cohorts of 6 dogs until either full BTK
occupancy was achieved, dose limiting toxicity (DLT) was identified, or if
18
objective tumor responses were noted in greater than 50% of a particular cohort.
The DLT was considered to be any grade 3 or 4 hematologic or non-hematologic toxicity based on established VCOG-CTCAE v1.1 criteria.83 Disease progression or clinical signs definitely related to disease were not considered adverse events
(AEs). The maximum tolerated dose (MTD) was considered to be one dose below that at which DLT occurred. Intrapatient dose escalation was permitted in dogs with a modified ECOG performance score of 0-1 in the face of disease progression or after day 14 pharmacokinetic assessment had been completed.84,85 Plasma samples were obtained for pharmacokinetic analysis and peripheral blood mononuclear cells (PBMCs) and lymph node FNAs were obtained for pharmacodynamic analysis.
Concomitant medications on clinical trial subjects
Supportive care was provided as needed to dogs while enrolled in this trial. Permitted concomitant medications included famotidine, omeprazole, maropitant and/or metronidazole. Prednisone was administered at a maximum dose of 0.5 mg/kg daily to treat tumor-related inappetence. Dogs on prednisone at enrollment were permitted to continue treatment if disease progression had occurred while on prednisone. Prednisone use was allowed during the course of the study in dogs with inappetence, regardless of prior prednisone use.
Additional supportive care was administered as clinically indicated. Intravenous pentobarbital injection was used for euthanasia, as elected by owner.
19
Pharmacokinetic analysis
Plasma samples were collected before dosing on days 0 and 14, and at
0.5, 1, 2, 4, 6, 8, 12 and 24 hours after dose administration on day 14. One
hundred microliters of heparinized blood was mixed with 600 µL of acetonitrile
and stored at -80°C within 20 minutes of collection. Plasma samples were
analyzed using a qualified HPLC-MS/MS method for acalabrutinib. Plasma
concentration-time data was analyzed by non-compartmental methods using
default settings and the NCA analysis object in Phoenix WinNonlin v6.3
(Pharsight, Mountain View, CA). Cmax/Dose and Tmax were determined following visual inspection of the plasma concentration versus time plots of individual patient data.
BTK occupancy analysis
Target occupancy analysis was performed on peripheral blood and fine needle aspirates from lymph nodes. Ninety-six-well Optiplate (Perkin Elmer,
Waltham, MA) plates were coated with 125 ng/well anti-BTK antibody (BD
Biosciences, San Jose, CA) and blocked with BSA (Sigma-Aldrich). A
biotinylated analogue of acalabrutinib was added to cell lysates to bind
unoccupied kinase sites on immobilized BTK. Cell lysates were then plated in
duplicate and incubated in presence or absence of 1 µM acalabrutinib to identify
minimal and maximal signals. Binding was detected by incubation with 10 ng/well
20
Streptavidin-HRP (Life Technologies), followed by ELISA Femto
Chemiluminescent Substrate (Thermo Fisher, Waltham, MA) and quantified on
an Envision plate reader (PerkinElmer).
Histology
Lymph node biopsies were fixed in 10% neutral buffered formalin for >24
hours, processed and embedded in paraffin, sectioned at 4 µm thickness, and stained with hematoxylin and eosin. Sections were evaluated and tumors histologically classified according to World Health Organization classification criteria by a board-certified veterinary pathologist.4
Response assessment
Up to five target lesions were measured using calipers, weekly during the
first four weeks on study, and every two weeks thereafter. Tumor response
assessments were performed using the Response Evaluation Criteria for
Peripheral Nodal Lymphoma in dogs (v1.0).86 A complete response (CR) was
defined as disappearance of all disease on two measurements separated by a
minimum period of 2 weeks. A partial response (PR) was defined as greater than
30% reduction in the sum of the longest diameters of target lesions documented
by two assessments separated by at least 2 weeks. Progressive disease (PD)
was defined as an increase of >20% in the sum of the longest diameters, using
the smallest sum since initiation of therapy as a reference, or appearance of any
21
new lesion(s). Stable disease (SD) was defined as the absence of criteria for either a response or progression. Objective response rate (ORR) was defined as the percentage of dogs with complete or partial responses. Progression free survival (PFS) was measured from day 0 until PD. Clinical benefit (CB) was defined as having SD, PR or CR for at least 28 days.
Adverse events (AEs)
AEs were graded in accordance with the VCOG-CTCAE v1.1 criteria;83 the
AEs reported here are restricted to those considered as possibly, probably or definitely related to acalabrutinib administration. For the patients that died during the trial, a peer-reviewed pathologic exam was performed to investigate cause of death.
Statistical analysis
For proliferation data, raw data were log transformed to reduce variance and skewness. Linear mixed effects models were applied to apoptosis data and the log-transformed proliferation data to account for the correlation of the observations from the same batch. A linear mixed effects model was used for analysis of the quantified western blots with dose dependent decrease (slope) in phosphorylation as the primary endpoint. Progression free survival (PFS) in dogs achieving PR versus SD was analyzed using log-rank test. P-value<0.05
22
was considered as significant. SAS 9.4 was used for those data analyses (SAS
Institute Inc., NC).
2.3 Results
Activity of acalabrutinib against canine lymphoma cells in vitro
Acalabrutinib antagonizes BCR activity
Herman and colleagues previously demonstrated inhibition of BTK and downstream targets in primary human CLL cells using ibrutinib.35 We
investigated whether similar pathway inhibition could be achieved using
acalabrutinib in canine lymphoma cells. The canine B-cell lymphoma line CLBL1
was treated with varying concentrations of acalabrutinib for 1 hour, and then
stimulated with anti-IgM and subjected to analysis of the BCR signaling pathway.
Dose-dependent inhibition of BTK autophosphorylation and phosphorylation of
downstream targets PLCG2, ERK, IKBA, AKT, and NFKB was observed at drug
concentrations between 10 nM and 1 µM (Figure 1A). To confirm the results
from CLBL1 cells, FNAs from 4 dogs with spontaneous B-cell lymphomas were
tested. These dogs, which were not included in the clinical trial, were either
treatment-naive or had received previous chemotherapy treatments. Treatment
and analysis were performed as with CLBL1 cells, and similar antagonism of
BCR signaling by acalabrutinib was observed regarding phosphorylation of BTK
and downstream targets (Figure 1B and 1C).
Acalabrutinib inhibits cell proliferation and survival 23
In CLBL1 cells treated with acalabrutinib, there was significantly
decreased cell proliferation compared to the vehicle control at concentration
1µM, and dose-dependent reductions in proliferation were observed for
concentrations up to 1µM. In addition, the cells showed a significantly reduced
trend of proliferation over the incubation time (Figure 1D and 1E). A reduced
percentage of viable cells over the tested drug concentrations (negative for both
PI and Annexin V, Figure 1F), was also observed following treatment with
acalabrutinib. These effects, though modest, are comparable with those
observed at physiologic concentrations in both primary CLL cells and DLBCL cell
lines. 36,87
The gene expression pattern of CLBL1 resembles the ABC subtype of canine
DLBCL
We next used GEP to sub-categorize the cell line CLBL1. Human DLBCL
cases can be sub-categorized into activated B cell (ABC)-like or germinal-center
B cell (GCB)-like subtypes on the basis of GEP.50 In human DLBCL, ibrutinib
treatment was more effective in patients with the ABC subtype (or non-GCB by
immunohistochemistry), compared to GCB DLBCL patients.88 Recently, canine
B-cell lymphomas were classified based on these subtypes, and prognostic differences (in PFS and OS) were similar to those observed with their human counterparts 50. We performed GEP analysis on CLBL1 to characterize it relative to ABC or GCB subtypes within canine B-cell lymphoma. Microarray data from
24
CLBL1 (published in GEO GSE43644) were analyzed using the RMA method,
and samples were clustered using centroid linkage. CLBL1 clustered with ABC
subtype samples from GSE43664 (Figure 2). Clustering was confirmed using
Euclidian distance, Pearson correlation, and un-centered correlation. With each method, CLBL1 was more similar to the ABC than to the GCB subtype.
Phase I study
Patient demographics
A dose-escalation study was initiated with acalabrutinib administered orally either once (QD) or twice daily (BID) at dosages between 2.5 mg/kg and 20 mg/kg in twenty companion dogs with spontaneously occurring B-cell lymphoma.
Patient demographics are summarized in Table 1. Peripheral lymph node biopsy specimens were morphologically subclassified as DLBCL with either immunoblastic or centroblastic cytologic features (Figures 3A and 3B). One sample was classified as diffuse large cell morphology with nodular architecture
(Figure 3C).
Pharmacokinetics and pharmacodynamics
Acalabrutinib absorption was delayed in most patients with the first
measurable levels of drug detected between 3 and 6 hours post administration
on day 14. Pharmacokinetic analysis was performed in a total of 7 dogs (2-3
dogs per cohort). The Cmax/Dose (mean±STD) was 195±94 ng/ml (419±201 nM),
and the median (range)Tmax 6 (3,8) hours . Twelve hour exposures (AUC0-12hr)
25
ranged from 3,086 to 31,445 hr*ng/ml with an average dose normalized AUC0-12hr of 1,005±476 hr*ng/ml. Pharmacokinetic data are summarized in Figure 4.
BTK target occupancy was evaluated in peripheral blood and lymphoma cells from FNAs. Full BTK occupancy (>90% of available target) was achieved in peripheral B-cells at 3 hours after dosing on day 1 with a dosage of 2.5 mg/kg
QD, for 5 of the 6 dogs in this cohort. A single dog with high peripheral B-cell count had 57% BTK occupancy on day 1, but had attained BTK occupancy of
94% prior to dosing on day 7. In samples taken at pre-dose on day 7 (i.e.,12 or
24 hours after prior dose administration), 83% to 99% BTK occupancy was observed among dogs in the 2.5 mg/kg QD cohort. A similar pattern was observed in higher dose cohorts, with complete BTK target coverage (Table 2).
Safety
Acalabrutinib was well tolerated by most patients, and maximum tolerated dose was not reached. Clinical observations related to disease, comorbid conditions, concomitant medications and research were not considered AEs. All clinical observations related to acalabrutinib were considered AEs and are listed in Table 3. Signs referable to the gastrointestinal system (anorexia, emesis, diarrhea and weight loss) and lethargy were the most commonly observed findings, regardless of attribution to research or to study drug. Gastrointestinal
AEs were responsive to medical management or were self-limiting. One dog was de-escalated from 10 mg/kg BID to 10 mg/kg QD dosing due to gastrointestinal
AEs.
26
Two dogs experienced severe adverse events during the study, both of
which were considered unlikely to be related to acalabrutinib administration.
Patient 17 was euthanized one week after initiation of treatment due to septic
shock in the face of severe disease progression. The cause of sepsis was not
identified. Patient 18 was hospitalized on day 3 for bacterial prostatitis and
emphysematous cystitis, resulting in immediate discontinuation of dosing with
acalabrutinib. This patient was euthanized on day 11. Necropsy of both patients
revealed no evidence of toxicity attributable acalabrutinib.
Response to therapy
Most dogs experienced a reduction in target lesion (lymph node) size from
baseline, as shown in Figure 5. Responses are summarized in Table 4A.
Clinical benefit (PR or SD ≥28 days) was observed in 30% of patients. Median
PFS for all patients was 22.5 days (Figure 6A). There was little variability in PFS when patients were stratified on relapse status at time of enrollment (Table 4B).
However, dogs achieving PR had significantly longer PFS (56 days) than those with SD (22.5 days) (Table 4C, Figure 6B). Median durations of response (DoR) for dogs achieving PR or SD were 49 days and 21 days, respectively. In addition, some dogs with disease progression were escalated to higher doses of acalabrutinib either QD or BID. Notably, dose escalation resulted in renewed or sustained clinical benefit in five of six dogs.
27
2.4 Discussion
BTK is a vital component of BCR signaling, and targeting BTK is clinically
efficacious in several hematologic malignancies, including B-cell lymphomas.88–91
Although treatment with ibrutinib has demonstrated durable responses in some
B-cell malignancies, notably CLL, ibrutinib has off-target activities that may limit
the potential uses of this therapeutic. For example, ibrutinib irreversibly inhibits
interleukin-2 inducible T-cell kinase39, which mediates signaling essential for NK- cell antibody dependent cellular cytotoxicity (ADCC).92 It has been reported that
concurrent administration of ibrutinib antagonizes rituximab-mediated ADCC in
vitro and in a murine lymphoma model93, and thus, combination of ibrutinib with monoclonal antibodies may not result in optimal efficacy. Additionally, the epidermal growth factor family receptors can be inhibited by ibrutinib.40 Some common adverse events observed with ibrutinib treatment include skin rash and gastrointestinal effects, including diarrhea, vomiting, and anorexia. Similar toxicities are observed with epidermal growth factor receptor inhibitors.94,95
Therefore, BTK inhibitors with greater target specificity could prove beneficial for patients if they result in fewer off-target effects.
As large animal models of lymphoma are scarce, we elected to use a canine model to evaluate safety and efficacy of acalabrutinib. Spontaneous cancers in dogs recapitulate their human counterparts with respect to biology, clinical behavior, molecular profiles, and conserved cytogenetic abnormalities, in addition to response and resistance to therapy.96 As tumors develop naturally in
28
animals with competent immune systems, the effects of novel agents on tumor
growth and metastasis can be modeled more accurately in dogs, compared with
mouse xenograft studies. Additionally, in spontaneously occurring cancer in
dogs, the tolerability and efficacy of novel agents can be evaluated in an outbred
population of dogs of various ages and co-morbidities. There are many examples where leveraging the dog model in preclinical drug development has informed the development path by providing pharmacokinetic/pharmacodynamic information, establishing dosing regimens, validating targets and identifying adverse events, and ultimately informing subsequent clinical studies in people.34,60,61
Additional support for canine B-cell lymphoma as a model of human
DLBCL comes from its classification into ABC and GCB subtypes43 and the
clustering of GEP from the canine CLBL1 cell line with ABC subtype of DLBCL.
These fundamental similarities between canine and human lymphoma
underscore the utility of comparative and translational research, providing the
opportunity to target biochemical pathways critical to lymphoma pathogenesis
and allowing preclinical assessment of novel therapeutics in a spontaneously
occurring disease model.
Our in vitro results show that BCR signaling activates BTK and downstream targets in canine lymphoma cells, and inhibition of this signaling decreases proliferation and survival of CLBL1 cells. This response to BTK
29
inhibition is similar to that demonstrated with ibrutinib in many types of human
NHL.35,97–99
We evaluated the safety and efficacy of acalabrutinib in companion dogs of various genetic backgrounds with spontaneous B-cell lymphoma. Acalabrutinib pharmacokinetics following oral dosing in dogs were sufficient to achieve plasma concentrations expected to elicit the desired anti-BTK pharmacodynamic effects.
Oral doses up to 15 mg/kg BID were well-tolerated, and biologic activity was observed in all cohorts. Full BTK occupancy was observed 3 hours after dosing in peripheral blood B-cells and FNA lymphoma samples obtained from all cohorts. These results demonstrated rapid tumor penetration by acalabrutinib.
Trough (pre-dose) BTK occupancy evaluated in peripheral blood B-cells and lymphoma FNAs on study day 7 remained high, reflecting steady state inhibition of the pathway. The ability to obtain matching tissue and blood samples at multiple timepoints is one advantage of using this dog model.
Clinical benefit was observed in 30% of patients. Notably, dogs with either treatment-naïve or drug-resistant NHL experienced clinical benefit. The administration of prednisone to 16 patients represents one potentially confounding variable to the clinical benefit and response data. However, 10 of 16 dogs had experienced disease progression even with concomitant prednisone administration prior to study enrollment, detracting from the potential anti- neoplastic benefits these patients may have received from prednisone.
Interestingly, this subset of 10 patients accounts for three of the most
30
pronounced and durable responses. It is also interesting that in nearly all dogs
that had dose escalations, additional clinical benefit was observed. This finding
suggests clinical benefit may be dose-dependent, and because acalabrutinib was
well-tolerated, higher doses may be better suited to use in follow up studies.
Results of the current study are comparable with early experiments with
ibrutinib, in which a small cohort of dogs with spontaneous B-cell lymphoma was
treated with dosages of 2.5 to 20 mg/kg QD. Twenty percent(1/5) of the dogs
with DLBCL achieved a PR in the ibrutinib studies 34, compared with 25% in the present study. ORR in the present study is also comparable with that observed in ibrutinib-treated DLBCL patients 38 (both are 25%), a finding which lends
additional support to the translational relevance of this model.
PFS appears abbreviated in this study (22.5 days). Unlike human CLL,
canine lymphoma is a rapidly progressive malignancy with median OS of only 4-6
weeks in newly-diagnosed patients without treatment. It is also important to note
that two-thirds of dogs were chemotherapy refractory, and survival in these
relapsed patients is often as short as 28 days, even with aggressive cytotoxic
regimens (reviewed in 100). Furthermore, early stage (stage 1) patients were excluded from the study. Taking this into consideration, ORR and PFS are more favorable than they appear at first glance. Additionally, the data suggest that this therapy may work best in a subset of dogs (the 5 dogs achieving PR).
Preliminary studies in human DLBCL suggest factors such as molecular subtype
(ABC vs. GCB) as well as specific mutations (MYD88, CD79, CARD11 and
31
TNFAIP3) may predict response to ibrutinib.38,88 We are currently pursuing additional studies to determine whether such molecular differences exist between our canine patients achieving a PR and those with SD or PD.
The toxicity profile of acalabrutinib is favorable in comparison to standard chemotherapy regimens often used for canine lymphoma. The most common
AEs were mild and gastrointestinal in nature (anorexia, vomiting) and responded to medical interventions or temporary drug discontinuation. Adverse events occurring in dogs treated with ibrutinib have not yet been described, and therefore, it is unknown how toxicities of acalabrutinib compare with those of ibrutinib. Therapy with cyclophosphamide, doxorubicin, vincristine, and prednisone combinations yields frequent and occasionally severe toxicities in dogs. Whereas this study focused on acalabrutinib monotherapy, the favorable
AE profile of acalabrutinib suggests combination strategies may support reduced doses of standard chemotherapeutic agents. Combination with other targeted therapies could yield an increase in response rates or durations. Such therapeutic strategies may help alleviate AEs associated with chemotherapy, in addition to improving responses and progression free survival.
This work demonstrates histologic, biologic and molecular similarities between canine and human NHL, including classification of the ABC molecular subtype of CLBL1 cells. It also reproduces the expected biologic responses to
BTK inhibition in vitro and in vivo with canine B-cell lymphoma. The value of spontaneously occurring canine lymphoma as a model for human DLBCL is
32
demonstrated. Using this model, acalabrutinib was orally administered at doses and schedules that provided important information about biologic action, tolerability, and anti-tumor efficacy in an aggressive disease setting. Based on these promising monotherapy results, further evaluation of acalabrutinib in combination with other agents in this spontaneous B-cell lymphoma model is merited.
33
2.5 Figures and Tables
Figure 1. Effects of acalabrutinib inhibitors on canine lymphoma cells.
Dose-dependent inhibition of BTK autophosphorylation (Y223), in addition to downstream targets, was observed via immunoblot at drug concentrations as low
(continued on the next page) 34
(Figure 1 continued)
as 0.01µM following 1 hour of treatment with acalabrutinib in the canine B-cell lymphoma CLBL1 cell line (representative of 3 repetitions) (A) and primary canine lymphoma cells treated ex vivo (representative of 4 patients, separate from the clinical study population) (B). C. Densitometry quantification of the western blots from B. Bands of phospho-proteins are normalized to respective total proteins and loading control. There was a significant dose-dependent decrease in phosphorylation for p-ERK (P=0.0028) and p-AKT (p=0.0019). D. Dose-dependent reductions in cell proliferation following daily treatment with acalabrutinib in the canine CLBL1 B-cell lymphoma cell line. Results are the mean of 5 independent experiments. Raw data were log transformed to reduce variance and skewness. Linear mixed effects models were applied to apoptosis data and the log- transformed proliferation data to account for the correlation of the observations from the same batch. p=0.012 E. Representative histograms showing a dose- dependent reduction in Edu incorporation from a single day at the 72 hour timepoint. F. Dose-dependent trend toward reductions in cell viability in CLBL1. Results are the mean of 3 independent experiments. Effects not statistically significant in a linear mixed effects model.
35
Figure 2. CLBL1 clusters with ABC-like canine lymphoma subtype. Two-way hierarchical clustering that combines CLBL1 with canine samples from GSE43664. In the colorbar at the top, CLBL1 is pink, and the samples from GSE43664 are blue for group 1 (GCB-like) and brown for group 2 (ABC-like).
36
Figure 3. Histopathology of peripheral lymph node biopsies from dogs enrolled in the acalabrutinib clinical trial. All patients were morphologically classified as DLBCL with either centroblastic (A) or immunoblastic (B) morphology. A single patient was noted to have marked nodular architecture reminiscent of follicular structures (C). (Hematoxylin and eosin)
37
Figure 4. Pharmacokinetic data. Plasma levels of acalabrutinib were measured at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after oral dose administration from 7 patients on day 14.
38
Figure 5. Reduced target lesion size in acalabrutinib treated dogs.
Waterfall plot showing percentage decrease in mean sum of longest diameter of index lymph nodes as compared to baseline measurements.
39
Figure 6. PFS. Kaplan-Meier curves showing overall PFS (A) and PFS of dogs achieving SD compared with PR (B). Dogs achieving PR survived significantly longer than those achieving SD (p=0.010).
40
ACP-196 (N=20) Mean 6.25 Age (Years) Median 5.5 Range 3-13 Pure Breed 14 Breed Mixed Breed 6 Male Castrated 7 Male Intact 1 Sex Female Spayed 12 Female Intact 0 Mean 24.74 Weight (kg) Median 20.7 Range 7.7-51.8 Yes 16 Prednisone No 4 Yes 14 Prior Chemotherapeutics No 6 DLBCL - IB 8 Lymphoma Subtype DLBCL – CB 10 Not Available 2
Table 1. Patient Demographics.
41
BTK dose and Peripheral B cells Fine Needle Aspirates PK Time point Day 1 3h Day 7 predose Day 1 3h Day 7 predose 93 90 2.5 mg/kg QD ND ND (n=6) (n=5) 99 92 100 82 10 mg/kg BID (n=2) (n=2) (n=2) (n=2) 97 96 95 84 15 mg/kg BID (n=4) (n=2) (n=3) (n=1) 91 93 98 85 20 mg/kg QD (n=3) (n=2) (n=3) (n=2)
Table 2. Percentage BTK target occupancy in peripheral blood B-cells and lymphoma aspirates of dogs following treatment with acalabrutinib.
All values are presented as the average percentage BTK occupancy relative to control levels of BTK in matched pre-study samples. Due to poor sample quality the 5 mg/kg cohort was not included in the analysis. ND – not determined.
42
Adverse Events (number of events by grade) Dose Weight Group Anorexia Diarrhea Lethargy Vomiting Nausea Seborrhea Loss 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 1 (n=6) 2
(n=5) 3 1 1 1 1 (n=2) 4 1 1 (n=3) 5 2 1 1 1 1 1 (n=4)
Table 3. Adverse events listed by grade and frequency.
43
A Be st Objective Dose Group Study ID Histologic Subtype PFS Response Response
1 DLBCL -0.55% SD 14 2 DLBCL-CB -1.60% SD 24 3# DLCBL-CB -38.01% PR 105 2.5 mg/kg QD 4 DLCBL-IB -8.06% SD 26 # 5 DLCBL-CB -35.90% PR 42 6 DLCBL-CB -0.88% SD 28 # 7 DLBCL-CB -7.54% SD 42 8 DLBCL-IB -5.05% SD 35 5 mg/kg QD 9 DLBCL -9.85% SD 21 # 10 DLBCL-IB -23.81% SD 49 # 11 DLCBL-CB -9.42% SD 7 12# DLCBL-CB -49.23% PR 56 10 mg/kg BID 20 DLCBL-CB -7% SD 18 14 DLCBL-CB -22% SD 14 20 mg/kg QD 15 DLBCL-IB 13% PD 6 16 DLBCL-IB -33% PR 16 + 17 DLBCL-IB -33.10% PD 7 18 DLCBL-CB -10% SD 11 15 mg/kg BID 21 DLBCL-IB -46% PR 70 24 DLBCL-IB 17.72% PD 7
Median Objective B PFS Response All patients 22.5 5 PR, 12 SD, 3 PD Relapsed 19.5 4 PR, 7 SD,1 PD Treatment Naïve 25 1 PR, 6 SD, 1 PD
Patient Median Median DoR C Response PFS PR 56* 49 SD 22.5* 21
Table 4. Clinical Response Rates.
(continued on the next page) 44
(Table 4 continued)
A. Patient response separated by treatment cohort is shown. Best response to treatment indicates the greatest percent reduction in mean sum of target lesions. Objective response and PFS are also shown. (PFS is measured in days) B. Median PFS and objective response for relapsed and treatment naïve groups. C. Median PFS and DoR for patients with PR and SD. *p=0.010 #This response was attained after dose escalation for some dogs P These patients were administered prednisone prior to enrollment and during the trial. p These patients began prednisone during the trial. +This patient is classified as PD due to progression in non-target organs identified at necropsy.
45
Chapter 3. Preclinical Evaluation of Acalabrutinib (ACP-196) in Chronic Lymphocytic Leukemia
3.1 Introduction
CLL is the most prevalent leukemia among adults. Although
chemoimmunotherapy may prolong the duration of remission and overall survival
among most patients with CLL, relapse virtually always occurs.101,102 This has prompted aggressive discovery efforts for new therapies in CLL. Because B-cell receptor (BCR) signaling is a driving factor for CLL tumor cell survival103,104,
proximal kinases involved in this pathway have become therapeutic targets. BTK
is immediately downstream of the B-cell receptor and is essential for the
activation of several tumor cell survival pathways relevant to CLL.105 In addition,
BTK is involved in chemokine-mediated homing and adhesion of CLL cells to the
microenvironment, which contribute to their maintenance and proliferation97,106. In
mice and humans, loss of BTK function results in a B-cell–dysfunction phenotype
with decreased serum immunoglobulin levels and an increased predisposition to
infections.107–109 The unique structure of BTK, which is characterized by a
cysteine (C481) within the ATP-binding pocket, makes it an attractive therapeutic
target. Ibrutinib is a first-in-class, irreversible small-molecule inhibitor of BTK that
has the ability to covalently bind to C481.34 Ibrutinib has shown substantial
single-agent activity in patients with relapsed CLL and in previously untreated
patients.90,110,111 Progressive disease during ibrutinib treatment is uncommon in patients with previously untreated CLL and also in patients with low-risk genomic
46
abnormalities.90,110,111 Among those with high-risk genomic features, progression occurs more frequently, either shortly after the start of ibrutinib therapy, owing to
Richter’s transformation (CLL that has evolved into large-cell lymphoma), or later with progressive CLL.112 Ibrutinib also irreversibly binds to other kinases (e.g.,
EGFR, TEC, ITK).34 These pharmacodynamics features may be responsible for ibrutinib-related adverse events that are not typically observed in BTK-deficient patients, such as rash, diarrhea, arthralgias or myalgias, atrial fibrillation, ecchymosis, and major hemorrhage.90,110,111 Importantly, side effects represent the most common reason that patients discontinue ibrutinib treatment.3,113
Acalabrutinib (ACP-196) is a second-generation, selective, irreversible inhibitor of
BTK that has improved pharmacologic features, including favorable plasma exposure, rapid oral absorption, a short half-life, and the absence of irreversible
targeting to alternative kinases, such as EGFR and ITK. Given the success of
ibrutinib in the treatment of relapsed CLL, we sought to evaluate the activity and
target specificity of acalabrutinib in vitro.
3.2 Materials and Methods
Immunoblotting
Primary CLL cells were isolated using RosetteSep™ Human B Cell
Enrichment Cocktail (Cat. #15064; Stemcell Technologies; Vancouver, BC,
Canada) followed by Ficoll Paque Plus™ density separation method (G.E.
Healthcare Biosciences AB; Uppsala, Sweden) per manufacturer instructions.
Primary T cells were isolated in a similar fashion using RosetteSep™ Human T 47
Cell Enrichment Cocktail (Cat . #15061; Stemcell Technologies). All primary cells
and cell lines were treated with indicated concentrations of acalabrutinib or
ibrutinib (Acorn Pharma Tech, LLC), washed twice with phosphate-buffered
saline (PBS), then replenished with RPMI media supplemented with 10% fetal
bovine serum prior to stimulation (RPMI/10% FBS). Primary CLL cells were then
stimulated for 15 minutes with plate-bound antihuman IgM (MP Biomedicals;
Santa Ana, CA). Plates were prepared by incubating anti-IgM in PBS at 10 µg/mL
overnight at 4°C. Wells were washed with PBS once immediately prior to plating
cells. Primary T cells and Jurkat cells were stimulated using plate-bound anti-
human CD3 antibody (Cat. #16-0037-85; eBiosciences; San Diego, CA)
prepared in a similar fashion to anti-IgM and soluble anti-human CD28 antibody
at 1 µg/mL (Cat. #16-0288; eBioscience) for 45 minutes. H460 cells were
stimulated with soluble epidermal growth factor at 50 ng/mL (Cat. #PHG011;
ThermoFisher Scientific; Grand Island, NY) for 10 minutes. Whole cell lysates
were prepared immediately after stimulation. Proteins were detected using the
following antibodies: anti-phospho-IKBA (Ser32, Cat. #2859), anti-IKBA (Cat.
#4812), anti-phospho-ERK1/2 (Thr202/Tyr204, Cat. #9101), anti-ERK1/2 (Cat.
#9102), anti-phospho-AKT (Thr308, Cat. #9257), and anti-AKT (Cat. #9272), anti-phospho-EGFR (Tyr1173, Cat. #4407) anti-phospho-EGFR (Tyr1068, Cat.
#2234), anti-EGFR (Cat. #2646), anti-JunB (Cat. #3753), and anti-NFAT (Cat.
#4389) (Cell Signaling Technologies; Danvers, MA).
48
Migration Assay
To test whether acalabrutinib inhibits chemokine mediated migration,
transwell plates (Sigma-Corning, Cat. #3414; Tewksbury, MA) containing 5uM porous inserts were used to separate the primary CLL cells from chemokine infused media. Cells were pretreated with either ibrutinib or acalabrutinib, plated in combination with either CXCL12 @ 100ng/mL or CXCL13 @ 500ng/mL (Cat.
#P48061 and #Q53X90, R&D Systems; Minneapolis, MN), and allowed to migrate for 3 hours at 37°C and 5% CO2. The number cells that had migrated
through the insert was then counted for 60 seconds at a constant rate on a
Beckman Coulter FC500 flow cytometer. The proportion of migrating cells was
normalized to a vehicle condition, and total input cell number for each treatment.
Chromium Release Assay
To evaluate the effects of acalabrutinib on NK cell mediated antibody
dependent cell cytotoxicity, a chromium release assay was performed using
isolated CLL cells from the peripheral blood of patients as previously
described.114 Briefly, 51Cr-labelled CLL cells and NK cells were incubated either
in media or with obinutuzumab (anti-CD52 monoclonal antibody) or trastuzumab
(anti-HER2 monoclonal antibody, negative control). Cells were washed prior to
combining culture at an effector to target ratio of 1:6. Following a 4-hour
incubation period, release of gamma radiation into the supernatant was
measured using a gamma counter. Percentage of cell lysis is determined by
49
normalizing values to CLL cells incubated in absence of effector cells
(spontaneous 51Cr release) and sonicated cells (maximal amount of 51Cr release)
as follows: 100 x (Experimental sample release – Spontaneous release) /
(Maximal release – Spontaneous release).
Viability Assay
Cell viability was measured using annexin-V/PI flow cytometry (Beckman-
Coulter; Miami, FL. Staining and analysis were performed as previously described by our laboratory.17
Statistical analysis
Linear mixed effects models were used for analysis of apoptosis and
chromium release assays to take account of the correlation among observations
from the same patient. The migration assay was analyzed using a paired two-
tailed T-test.
3.3 Results
Acalabrutinib is a potent and specific inhibitor of BTK
Although ibrutinib inhibits BTK phosphorylation by Western blot at
nanomolar doses and also in vivo in murine models, it also inhibits numerous
other unfavorable targets34, and to this effect, acalabrutinib was developed as a more potent and specific inhibitor of BTK. To confirm that acalabrutinib can irreversibly inhibit BCR signaling, we performed immunoblotting using primary 50
patient CLL cells (Figure 7A). Cells are treated with acalabrutinib, copiously
washed, then stimulated with anti-IgM to activate the B-cell receptor (BCR), Our
data show that downstream targets of activated, phosphorylated BTK, including
phospho-ERK, phospho-IκB-α, and phospho-AKT are all inhibited at
concentrations of acalabrutinib as low of 10nM, while total amount of each
protein remains unchanged (N=6). Thus, BCR signaling was potently inhibited by
this compound.
Activity of ibrutinib on the receptor tyrosine kinase EGFR is speculated to
result in the adverse events of rash and diarrhea in patients.40 To determine whether acalabrutinib has off-target effects on this molecule, we treated H460 lung cancer cell line with either ibrutinib or acalabrutinib at the indicated concentrations followed by stimulating cells through EGFR using soluble EGF
(Figure 7B). While ibrutinib shows potent and dose-dependent inhibition of
EGFR phosphorylation at 2 different sites, acalabrutinib did not affect the phosphorylation of this target, thus, supporting the specificity of acalabrutinib.
Ibrutinib is also a potent inhibitor of ITK39, a molecule which is highly
important to T-cell and NK cell function, and inhibition of this molecule leads to
reduced antibody dependent cell cytotoxicity (ADCC).93,115. ADCC is an important
mechanism of action for monoclonal antibodies, including rituximab and
obinutuzumab, which are a component of young and old adult CLL therapy
(reviewed in 116). Clinical trials using ibrutinib in combination with rituximab do not show improved outcomes when compared with ibrutinib monotherapy, further
51
supporting the concept that ibrutinib may reduce activity of monoclonal
antibodies.117 To test the effects of acalabrutinib of T-cell receptor signaling mediated by ITK, we treated both Jurkat cells, a T-cell leukemia cell line, and primary healthy human T-cells with ibrutinib or acalabrutinib, followed by dual stimulation through the T-cell receptor with anti-CD28 and anti-CD3 antibody ligation (Figures 7C and 7D). With the Jurkat cell line, ibrutinib reduced concentration of the transcription factors JunB and NFAT in addition to phosphorylated IκBα, while acalabrutinib showed no inhibition of these targets.
Using primary T-cells, nuclear and cytoplasmic lysates were evaluated separately. After ibrutinib treatment but not acalabrutinib treatment, the nuclear fraction showed reduced translocation of JunB and NFAT, while the cytoplasmic fraction showed reduced phosphorylation of IκBα.
Targeting BTK with acalabrutinib leads to modest reduction in cell viability but potent inhibition of chemokine-driven migration
Unlike traditional chemotherapeutic regimens which are potent inducers of tumor cell death, BTK inhibitors appear to function by alternative means. De
Rooij, et al. previously have shown that ibrutinib treatment reduces CLL cell migration in response to the chemokines CXCL-12 and CXCL-13, both of which are important in homing of CLL cells to proliferation centers in the lymph node.106
To confirm that acalabrutinib also inhibits chemotactic migration, cells treated
with either vehicle, acalabrutinib or ibrutinib were plated and allowed to migrate
52
through a porous membrane towards chemokine infused media (Figure 8). Both acalabrutinib (p=0.002 and p=0.002) and ibrutinib (p=0.01 and p=0.003) reduced chemtotactic migration in response to CXCL12 and CXCL13, supporting a similar mechanism of action of both compounds and confirming the BTK-dependence of this phenomenon. Annexin-V/PI of primary patient CLL cells treated with acalabrutinib at varying doses for 24, 48 or 72 hours indicates that, much like ibrutinib, acalabrutinib induces modest dose- and time-dependent apoptosis
(Figure 9).
Acalabrutinib does not inhibit NK cell driven ADCC
Previously, it has been shown that ADCC mediated by NK cells is highly dependent on the signaling molecule ITK, and indeed, clinical trials of ibrutinib in combination with rituximab have yielded no additive benefit when compared with ibrutinib monotherapy.117 Given the lack of off-target effects of acalabrutinib on
ITK, we hypothesized that acalabrutinib would lack adverse effects on in vitro cytolysis of CLL cells by allogenic healthy donor NK cells. CLL cells loaded with radioactive chromium and NK cells were treated with either ibrutinib or acalabrutinib, and co-cultured in the presence of the anti-CD20 monoclonal antibody obinutuzumab, no antibody or the HER2 monoclonal antibody trastuzumab (negative control) (Figure 10). Chromium release was then measured using a gamma counter. Our data show that while ibrutinib inhibited
53
cytolysis of CLL cells in the presence of active antibody directed at CD20,
acalabrutinib had no effect on this process.
3.4 Discussion
BTK inhibitor therapy has revolutionized treatment of CLL, and other B-cell
cancers. Ibrutinib is a first in class BTK-inhibitor with potent on target activity in
CLL, but therapy with this drug can result in adverse events, causing some
patients to discontinue treatment.90,110–112 These side effects may be related to
ibrutinib’s activity on alternative kinases that are present in non-cancerous
tissues. Acalabrutinib is a more specific inhibitor of BTK. Our data confirm that
acalabrutinib is a potent inhibitor of BTK and BCR signaling. It induces mild
apoptosis, comparable to previous publications with ibrutinib35, but more importantly, it robustly inhibits chemotactic migration, a phenomenon important for homing to proliferation centers in the microenvironment.106
Acalabrutinib is a highly specific BTK inhibitor, and has no effect on the
kinases EGFR, which is highly expressed in epithelial cells, and ITK, which is
important for signaling in T-cells and NK cells. The lack of activity on EGFR may
reduce incidence of cutaneous rash and diarrhea, and a head-to-head trial of
acalabrutinib vs. ibrutinib would confirm this. Indeed, early clinical data using
acalabrutinib have shown a very low incidence of these adverse events with
grade 3-4 diarrhea and rash occurring in no more than 2% of patients.41 ITK is important for activation of T-cells and NK cells.39,93 Activity of ibrutinib against ITK
54
results in reduced rituximab-mediated ADCC in vitro93, and in clinical trials, the
addition of rituximab to an ibrutinib regimen does not result in improved patient
responses.117 Acalabrutinib lacks activity against ITK, and, thus, may be more amenable to combination therapy with monoclonal antibodies, such as obinutuzumab, shown in this manuscript.
Overall, these data suggest that acalabrutinib may improve upon the
established success of first in class BTK inhibitors by reducing treatment-related
adverse events. Phase I clinical trial data using acalabrutinib in relapsed and
refractory CLL suggest reduced incidence of grade 3-4 adverse events, such as
diarrhea and rash, among others, though a head-to-head clinical trial is
necessary to validate this finding.41 These early clinical data also indicate robust activity comparable to or improving upon that observed with ibrutinib. This finding is imperative, as the pan-kinase inhibitory characteristics of ibrutinib have led to speculation in the scientific community regarding the importance of multi-kinase inhibition in achieving clinical response. The phase I clinical trial data and our in
vitro assays for apoptosis and chemotactic migration refute this concept and
confirm that BTK is, indeed, the key kinase whose inhibition results in clinical
response. Taken as a whole, this manuscript confirms the importance of BTK in
CLL and encourages the continued development of the specific BTK inhibitor
acalabrutinib for the treatment of CLL and other B-cell malignancies.
55
3.5 Figures and Tables
Figure 7. Acalabrutinib (ACP-196) is a specific inhibitor of BTK. A. Western blot showing dose-dependent inhibition of signaling molecules downstream of BTK in primary patient CLL cells. Cells are treated with ACP-196, washed, and then stimulated with plate-bound anti-IgM. Phosphorylated products of ERK, IkBa and AKT are decreased following treatment with ACP-196, while total concentration of these proteins remains constant. (Representative blot from 7 patients.) B. Western blots showing that ACP-196 does not inhibit EGFR autophosphorylation in H460 lung cancer cells, while ibrutinib produces a dose- dependent inhibition of this molecule. Cells were pretreated with ACP-196 or ibrutinib followed by washout, then stimulation with soluble EGF. (Representative blot from 3 repetitions.) C and D. Western blots showing TCR signal transducers downstream of ITK in Jurkat cells (C) and primary T-cells (D). Briefly, cells are pretreated with ACP-196 or ibrutinib followed by washout and stimulation with plate-bound anti-CD3 and soluble anti-CD28. ACP-196 does not inhibit ITK or
(continued on next page) 56
(continued from Figure 7)
TCR signaling. Ibrutinib induces potent dose-dependent inhibition of molecules downstream of ITK, including transcription factors JunB and NFAT and pIkBa. Lamin B and actin are negative controls for cytoplasmic and nuclear lysate purity, respectively. (Representative blots from 3 experiments each.)
57
p=0.002
M edia 15 p=0.01 CXCL12
p=0.002 CXCL13 10 p=0.003
5 Fold M igration
0
Vehicle Vehicle Vehicle
Ibrutinib 1uM Ibrutinib 1uM Ibrutinib 1uM
Acalabrutinib 1uM Acalabrutinib 1uM Acalabrutinib 1uM
Figure 8. Chemokine dependent migration of primary CLL cells treated with acalabrutinib.
Migration across the transwell filters is increased in response to chemokines CXCL12 and CXCL13, and this migration is partially abrogated by the addition of acalabrutinib or ibrutinib. N=11 patients.
58
Figure 9. Acalabrutinib (ACP-196) induces modest apoptosis in CLL cells.
Cell viability was assessed after 24, 48 and 72 hours in CLL cells treated with either acalabrutinib at indicated, vehicle, or fludarabine (positive control). There is a modest reduction in cell viability which is both time and dose dependent in the acalabrutinib treatment conditions. Data are not statistically significant. N=7 patients.
59
100 Media Ibrutinib 80 Acalabrutinib
60 *
40 ADCC (%) ADCC
20
0
No antibody Trastuzumab Obinutuzumab
Figure 10. Acalabrutinib (ACP-196) does not inhibit NK cell mediated ADCC. Data are from a chromium release assay showing relative ADCC of primary CLL patient cells treated in vitro with the monoclonal antibody obinutuzumab in combination with ACP-196, ibrutinib or no inhibitor (media). CLL cells are loaded with chromium, NKL cells and CLL cells are treated with ACP-196 or ibrutinib, and cells are then co-incubated in the presence of the anti-CD20 antibody obinutuzumab, anti-HER2 antibody trastuzumab (negative control), or no antibody. Chromium release from cells treated with ACP-196 is similar to those with media only (no inhibitor). However, ibrutinib significantly reduced NK cell mediated ADCC. Effector:target ratio 25:1. *p<0.01
60
Chapter 4. Conclusions and Future Directions
Though less than a decade has passed since ibrutinib received FDA
approval, this therapeutic has shifted the treatment paradigm for CLL. Patients
that were once treated with toxic chemotherapeutics and bone marrow
transplants, therapies which result in significant morbidity and even death, can
now receive a daily pill with relatively low side effects and achieve durable
remission.110 Our data encourage the further development of more selective BTK
inhibitors to provide incremental improvements to this therapy. But in spite of this
success, resistance to BTK inhibitors presents an emerging problem, and future
efforts should also be directed toward the understanding of resistance in B-cell
cancers and the development of therapies to circumvent this problem.
4.1 Circumventing BTK Inhibitor Resistance in CLL
In CLL, acquired resistance occurs occasionally and is nearly always
attributed to a mutation in BTK, specifically C481S which prevents covalent
binding of ibrutinib. This mutation occurs with or without additional mutations in
downstream effectors of BTK, such as PLCγII.112,118,119 Small numbers of patients have shown other mutations in BTK, such as C481F/Y/R, T474I/S, and L528W, all of which affect the hydrophobic binding pocket where ibrutinib resides112, and
T316A which is predicted to affect BTK’s scaffolding function120. Since acalabrutinib binds to the same C481 residue as ibrutinib, acalabrutinib is not an
appropriate therapy for these relapsed patients. There are, however, a number of 61
therapeutics under development to circumvent this problem. ARQ-531, GDC-
0853, and SNS-062 are all reversible BTK inhibitors that function via competitive
inhibition of ATP. These compound bind to the ATP-binding pocket via
hydrophobic interactions and independent of the 481 amino acid residue and,
thus, have in vitro activity against the C481S mutant version of BTK.121–123 These
inhibitors could be administered as a frontline therapy, which would likely prevent
relapse in via C481S mutation, or after detection of the C481S mutant.
For CLL patients possessing activating mutations in PLCγII or alternative
BTK mutations (C481F/Y/R, T474I/S, and L528W), inhibition of BTK may no
longer be effective, and combination therapies to block alternative survival
pathways or other targets in the B-cell receptor should be examined. Indeed,
administration of these combination therapies upfront may prevent the
emergence of the elaborated mutations. An example of this approach involves
the novel class of compounds that inhibit MALT1, a member of the trimeric
protein complex located in the distal portion of the BCR cascade that serves to
activate NFκB signaling.124 Small molecule inhibitors are currently under
development and have shown preclinical activity in B-cell cancers, but protein
stability has precluded development of a viable therapeutic for clinical trials.125
Additionally, the potential for therapy-related adverse events should be considered. MALT1 is relatively specific for lymphoid cells, but is essential in activation of T-cells, which could lead to immunosuppressive side effects.126,127
62
Finally, there is a small subset of patients that exhibit primary resistance of unknown mechanism or resistance unrelated to molecular alterations in BCR signaling. In this latter group, deletions in 8p encompassing tumor necrosis factor-related apoptosis-inducing ligand-receptor (TRAIL-R), a caspase-activating receptor, have been identified in some patients128, and thus, abrogating this resistance to apoptosis makes sense. The anti-apoptotic protein BCL2 is upregulated in most cases of CLL owing to a chromosome 13q deletion leading to loss of negative regulators miR-15 and 16129, and as such, targeting BCL-2
with a small molecule inhibitor represents a logical therapeutic strategy. Early
clinical trials using the BCL-2 inhibitor venetoclax as a monotherapy in relapsed
CLL have shown high response rates and few adverse events130, and trials pairing venetoclax with ibrutinib are ongoing.
Combination with immunotherapies, including immune checkpoint blockade, represents another approach to enhance BTK inhibitor response. As an ITK inhibitor, ibrutinib selectively represses Th2 cell activity, leading to a Th1 predominant immune response.39 As the mechanism of action for immune
checkpoint blockade and chimeric antigen receptor T-cells (CARTs) depends on
T-cell activation, this Th1 selective pressure could further enhance CD8 and CD4
T-cell responses. Further supporting this strategy, CTLA-4 is overexpressed on
T-cells of CLL patients in comparison with healthy donors131, and PD-1 increases
on the T-cells of patients with progressive disease132. When expressed by the cancer cells, some immune checkpoints, such as CTLA-4, contribute to survival
63
and proliferation of the cancer cells themselves133. Arguing against immune
checkpoint blockade in combination with kinase inhibitors is the observation that
PD-L1 is downregulated on cancer cells after PI3K and BTK inhibitor treatment
(unpublished data).
4.2 Understanding and Circumventing BTK Inhibitor Resistance in Aggressive
Lymphoma
In contrast to CLL, mechanisms of BTK inhibitor resistance in aggressive lymphomas are complicated and poorly defined. Approximately 30% of MCL patients have primary resistance to ibrutinib134, and some studies have
associated this with either increased PI3K/AKT activity135, and/or loss of function
mutations in TRAF2 or BIRC3 leading to upregulated alternative NFκB signaling driven by NIK.136 Additionally, small numbers of patients have confirmed C481S mutation as in CLL.135,137 Recently, investigators have identified point mutations
in PIM1 and ErbB4, though the effect of these mutations was not
characterized.138The significance of this finding could be further defined through
computational protein modeling and investigation of functional protein alterations
in vitro using, for example, molecular cloning assays. A final alteration in MCL thought to play a role in BTK inhibitor resistance is the upregulation of
CD40:CD40L interaction in the microenvironment leading to increased alternative
NFκB signaling and abrogate the need for signaling through the BCR139, but this mechanism has not yet been validated in vivo with human patient samples.
64
Ibrutinib has been overall less effective as a therapy in DLBCL with an
ORR of 25% in relapsed/refractory patients.38 Patients with the activated B-cell
(ABC) subtype of DLBCL have a higher rate of response to ibrutinib than germinal center B-cell (GCB) patients. Other investigators have attempted to explain this phenomenon in relation to NFκB pathway addiction, which in ABC
DLBCL is driven by BCR or MYD88/toll-like receptor (TLR) signaling.140,141 BTK
is an integral signaling molecule in the BCR pathway and has also been shown
to be activated after CpG stimulation vial TLR9.29 Logically, it has been shown that patients devoid of activating mutations in this pathway or with mutations upstream of BTK (CD79) are BCR pathway addicted, making them sensitive to ibrutinib.38 On the other hand, patients bearing activating mutations downstream
of BTK , such as in CARD11 and/or MYD88, or inactivating mutations in
TNFAIP3 can drive NFκB activity in spite of BTK inhibition and are resistant to therapy.38 Though useful, these mutations unfortunately only predict response in a minority of patients; for example, only 40%, 33%, and 38% of patients with WT
MYD88, WT CARD11, and WT TNFAIP3 achieve CR or PR, respectively. Thus, resistance is still incompletely explained and represents an open avenue for investigation.
One approach to defining the mechanism of BTK inhibitor resistance in these aggressive B-cell cancers is to evaluate somatic mutations in search of oncogenic drivers that abrogate dependence on B-cell receptor signaling. This approach has already been attempted by multiple investigators described
65
above.38,136–138 However, the publications that identified TRAF2, BIRC3, PIM1
and ErbB4 aberrations in MCL and MYD88, CARD11, and TNFAIP3 in DLBCL all
applied targeted sequencing for these discoveries. This approach carries the advantage of increased sequencing depth, thus identifying low variant allele frequency mutations but sacrificing identification of novel somatic mutations which could play a role in resistance. As an alternative, whole exome sequencing could identify novel protein coding genes involved in resistance, and whole genome sequencing could be employed to include non-protein coding genes, such as micro-RNA. The disadvantages to these large scale genomic studies include the need for a large sample size to achieve statistical significance and difficulties with sequencing errors and data processing.
In our acalabrutinib canine studies, dogs exhibited similar response rates to human DLBCL patients treated with BTK inhibitors88, and thus, our canines may represent appropriate models for DLBCL resistance mechanisms. Whole exome or whole genome sequencing of lymph node samples from baseline to relapse could pinpoint somatic mutations involved in acalabrutinib resistance.
Given the prevalence of primary resistance in our population, it is likely the subclone/s responsible for resistance were already present at baseline and then grew out during treatment, so examination of variant allele frequency would be prudent. Given the relative paucity of published SNP databases for canines, analysis of all samples against a germline for each animal is essential. Once identified, functional prediction analysis and in vitro functional studies could
66
provide proof of principle that a given mutation contributes to enhanced survival
and BTK inhibitor resistance.
Gene set enrichment analysis via either microarray analysis or
transcriptome sequencing represents an alternative approach to defining ibrutinib
resistance. In these experiments, proprietary software are used to analyze large
scale data sets, identifying enrichment of mRNA from genes in certain pathways.
Samples can be compared from different niches (i.e. bone marrow vs. peripheral
blood) or from baseline to relapse in a given patient. It is plausible that resistance
to BTK inhibitors may be driven by upregulation of molecular pathways in
absence of somatic mutations. This technique would be especially useful to
identify signals from the microenvironment that are contributing to cell survival,
such as is described regarding CD40:CD40L interaction contributing to ibrutinib
resistance in MCL.139 Retrieving tumor samples from the microenvironment where cells receive proliferation signals, such as bone marrow or lymph node, is imperative for these studies.
One final avenue to identify resistance mechanisms involves the examination of cellular uptake, efflux, and detoxification of BTK inhibitors.
Though this represents a fervid topic in chemotherapeutics especially in the context of multi-drug resistance, surprisingly little investigation has revolved around kinase inhibitors including ibrutinib and acalabrutinib. ATP-binding cassette (ABC) transporters represent one class of drug efflux pumps with exceptional notoriety for mediating drug resistance and include P-gp, MRP1, and
67
BCRP (reviewed in 142). Classically, these efflux pumps confer resistance to a
variety of chemically dissimilar compounds, including vinca alkaloids, taxanes,
anthracyclines, calcium channel blockers, HIV protease inhibitors, and
epipodophyllotoxins. As most studies of tyrosine kinase inhibitor resistance have
focused on genetic mechanisms, there are limited reports involving the effects of
efflux pumps. Successful identification of ABC proteins involved in kinase
inhibitor efflux have occurred with imatinib (BCR-ABL inhibitor)143, nilotinib (BCR-
ABL inhibitor)144, dasatinib (SRC and BCR-ABL inhibitor)145, gefitinib (EGFR
inhibitor)146, erlotinib (EGFR inhibitor)147, lapatinib(EGFR and HER2 inhibitor)148, and sunitinib (PDGFR and VEGFR inhibitor)149. Though two studies postulate that ibrutinib is an inhibitor of ABC family proteins150,151, there are currently no published studies evaluating ability of efflux pumps to remove BTK inhibitors and contribute to resistance. The affinity and nature of interaction of kinase inhibitors with drug efflux pumps can be measured by ATP hydrolysis, which measure efflux pump activity, and abrogation of IAAP (125I) incorporation, indicating
incorporation of the drug into substrate-binding sites. Differential expression of pumps between relapsed and therapy naive patients can also be evaluated at the protein and mRNA level.
Clinically, combating resistance in B-cell lymphomas is similar to our approach to non-C481S ibrutinib-resistant CLL. Published data suggest growth and survival independent of the BCR in MCL and DLBCL, so alternative survival pathways should be targeted. MCL is prototypically driven by the overexpressed
68
oncogene cyclin D1 which complexes with cyclin dependent kinases 4 and 6
(CDK 4/6), leading to aberrant cell cycle progression.152 Clinical trials using palbociclib, an inhibitor of CDK 4/6, are underway and show great promise.
Unfortunately, CDKs represent a ubiquitously expressed target, and early clinical data indicate toxicities, specifically neutropenia, are dose-limiting with this compound.153 Other targets include BCL2, which is often overexpressed in
DLBCL and MCL just as in CLL, and NFκB, as activity of this pathway is
associated with ibrutinib resistance38,121. Over 700 agents are available to target
canonical and noncanonical NFκB signaling (reviewed in 154), but none have
received FDA approval, perhaps due to the ubiquitous target expression and
consequential adverse effects of treatment.
Over the past decade we have established the importance of BTK in B-cell
cancers and observed the tremendous success of inhibitors for this protein.
Unfortunately, as the number of patients receiving BTK inhibitors increases, we
observe increasing number of cases of primary and secondary resistance. This
represents an active area of research for many investigators. A slew of data
analyzing genes and pathways involved in this resistance has been published
over the course of just a few years, and these data are guiding future therapeutic
strategies which will either prevent the emergence of resistance or treat those
that have already developed resistance. Most clinical trials involving BTK inhibitor
resistant patients are in very early phases, and it remains to be seen whether our
69
molecular findings from these resistant patients have successfully directed therapies.
70
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