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Mechanistic and Preclinical Insights from Mouse Models of Hematologic Cancer Characterized by Hyperactive Ras

Anica Wandler1 and Kevin Shannon1,2

1Department of Pediatrics, Helen Diller Family Cancer Research Building, University of California, San Francisco, San Francisco, California 94158-9001 2Comprehensive Cancer Center, Helen Diller Family Cancer Research Building, University of California, San Francisco, San Francisco, California 94158-9001 Correspondence: [email protected]

RAS genes are mutated in 5%–40% of a spectrum of myeloid and lymphoid cancers with NRAS affected 2–3 times more often than KRAS. Genomic analysis indicates that RAS mutations generally occur as secondary events in leukemogenesis, but are integral to the disease phenotype. The tractable nature of the hematopoietic system has facilitated gener- ating accurate mouse models of hematologic malignancies characterized by hyperactive Ras signaling. These strains provide robust platforms for addressing how oncogenic Ras expres- sion perturbs proliferation, differentiation, and self-renewal programs in stem and progenitor cell populations, for testing potential therapies, and for investigating mechanisms of drug response and resistance. This review summarizes recent insights from key studies in mouse models of hematologic cancer that are broadly relevant for understanding Ras biology and for ongoing efforts to implement rational therapeutic strategies for cancers with oncogenic RAS mutations.

omatic RAS gene mutations were first iden- vating protein (GAP) that is a core component Stified in leukemia in the 1980s (reviewed in of the Ras/GAP molecular switch (reviewed in Bos 1989). Subsequent studies encompassing Cichowski and Jacks 2001), is also recurrently

www.perspectivesinmedicine.org thousands of hematologic cancers have defined mutated in a number of hematologic cancers the overall incidence of NRAS, KRAS, and (Ward et al. 2012). Wefirst summarize key find- HRAS mutations in different blood cancers, ings from studies of human patients that have the frequency of individual amino acid substi- informed our current understanding of the role tutions, the likely order in which these muta- of these mutations in blood cancers and refer tions are acquired during leukemogenesis, and readers to a recent review for additional back- the status of RAS mutations detected at diagno- ground information. sis, during disease remission, and after relapse The overall prevalence of RAS/NF1 muta- (reviewed in Ward et al. 2012). The NF1 tumor tions in hematologic malignancies ranges from suppressor gene, which encodes a GTPase acti- 5% in myelodysplastic syndrome (MDS) to

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A. Wandler and K. Shannon

20% in acute myeloid leukemia (AML) to cancers with KRAS, NRAS,orNF1 mutations, 40% in juvenile myelomonocytic leukemia we emphasize the use of these models as plat- (JMML). In contrast to most solid cancers, forms for investigating mechanisms of drug re- NRAS mutations predominate over KRAS mu- sponse and resistance that may inform efforts to tations by a ratio of 3:1 in hematologic ma- target oncogenic Ras signaling in both hemato- lignancies (Ward et al. 2012). Deep sequencing logic and nonhematologic cancers. of human tumors indicates that RAS and NF1 mutations do not initiate most hematologic cancers, but typically comprise secondary TRANSGENIC AND RETROVIRAL TRANSDUCTION/TRANSPLANTATION events that cooperate with antecedent muta- MODELS tions in genes encoding transcription factors and proteins that regulate epigenetic programs Oncogenic KRAS, NRAS, and HRAS mutations in hematopoietic stem and progenitor cells are distributed nonrandomly across different (HSPCs) (Jan et al. 2012; Shlush et al. 2014; cancers. The prevalence of NRAS and, to a lesser Lindsley et al. 2015). JMML, an aggressive can- extent, KRAS mutations in human myeloid ma- cer of young children, is a notable exception to lignancies prompted efforts to develop mouse this general rule (Jankowska et al. 2011; Saka- models by expressing different mutant Ras al- guchi et al. 2013; Stieglitz et al. 2015). Interest- leles under the control of various promoters. ingly, RAS (and FLT3) mutations identified at Transgenic animals in which the mouse mam- diagnosis are invariably undetectable in blood mary tumor virus (MMTV) long terminal re- and bone marrow samples from patients with peat (LTR) was used to drive oncogenic Nras AML and acute lymphoblastic leukemia (ALL) expression developed T- and B-cell lymphoblas- analyzed during remission (Lindsley et al. tic lymphomas and mammary carcinomas 2015). This contrasts with mutations in genes (Mangues et al. 1996). Using this same promot- such as DMNT3A, TET2, and IDH1/2, which er to drive transgenic expression of Hras caused frequently persist (Chou et al. 2012; Corces- B-cell lymphoblastic lymphomas at low fre- Zimmerman et al. 2014; Klco et al. 2015). Fur- quency. While disappointing at the time be- thermore, RAS mutations that are present at cause RAS gene mutations are more frequent diagnosis do not invariably reappear at disease in myeloid malignances and were not thought relapse, underscoring the impact of treatment- to contribute to lymphoid cancers, RAS and induced selective pressure on the dominant NF1 mutations were later identified in certain clone and highlighting the subsequent genetic aggressive types of ALL such as early T-cell pre- evolution that occurs in response to it. Finally, cursor ALL (ETP-ALL) and hypodiploid B lin- www.perspectivesinmedicine.org and importantly, when relapse follows a period eage ALL (Zhang et al. 2012; Holmfeldt et al. of remission in patients with acute leukemia, 2013). molecular analysis frequently reveals outgrowth A seminal study showed that retroviral of a rare clone with intrinsic drug resistance that transduction of murine bone marrow with the was already present at diagnosis (reviewed in BCR-ABL transgene, a potent upstream activa- Jan and Majeti 2013). tor of Ras and of the Raf/MEK/ERK effector The broad subject of mouse cancer models pathway (Sawyers 1999), followed by transplan- driven by oncogenic Ras genes is reviewed else- tation into irradiated recipient mice, caused where in the literature (Drosten et al. 2017; Jacks myeloproliferative neoplasms (MPNs) that 2017). Here we focus on mouse models of he- closely resembled human chronic myeloid leu- matologic malignancies characterized by RAS kemia (CML) (Daley et al. 1990). Following this and NF1 mutations, summarize key insights paradigm, a number of groups pursued a sim- from these systems, and discuss the advantages ilar strategy with RAS oncogenes (Fig. 1), which and potential liabilities of different experimen- was facilitated by development of the murine tal approaches. Importantly, as no mechanism- stem cell virus (MSCV) vector. In one study, based treatments exist for the 25% of human 60% of irradiated recipient mice injected

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Insights into Ras from Mouse Models of Hematologic Cancer

Transduce with GFP-tagged retroviral vectors

Harvest bone Harvest acute marrow cells leukemia cells

or Sort to obtain Transplant into Harvest fetal GFP+ cells irradiated mice liver cells

Figure 1. Retroviral transduction/transplantation to generate acute leukemias. Bone marrow or fetal liver cells are isolated from donor mice, transduced with retroviral vectors, sorted to obtain a pure population, and then transplanted into irradiated recipient mice. These animals develop acute leukemias that can be harvested and analyzed. GFP, Green fluorescent protein.

with bone marrow cells that had been trans- an invasive AML-like disease and no effect of duced with a retrovirus expressing an oncogenic viral titer was observed (Parikh et al. 2007). The Nras allele developed a spectrum of myeloid robust ability of oncogenic Hras to induce my- malignancies (MacKenzie et al. 1999). However, eloid malignancies despite the very low fre- the phenotypic variability, incomplete pene- quency of HRAS mutations in patient samples trance, and prolonged latency combined with (Ward et al. 2012), coupled with the observed both impaired in vitro proliferation and a high effects of viral titer, suggests that the super- rate of apoptosis in Nras-infected cells provided physiological levels of Ras expressed in these an early indication that levels of oncogenic Ras systems strongly influence disease phenotypes. expression strongly modulate cellular pheno- Importantly, the hematologic cancers that types. Consistent with this idea, positioning emerge from retroviral transduction/transplan- the Nras oncogene downstream of an internal tation experiments invariably exhibit clonal ribosomal entry site (IRES) in the MSCV retro- retroviral integrations (MacKenzie et al. 1999; viral backbone enhanced induction of MPNs Parikh et al. 2006, 2007), arguing that a specific and AML, which was likely caused by a lower level of oncogenic Ras protein expression is www.perspectivesinmedicine.org level of expression (Parikh et al. 2007). A direct strongly selected for, or that misregulation of comparison of the tumorigenic capacity of on- genes near the integration site or other cooper- cogenic Hras, Nras,orKras was performed by ating somatic mutations are required to induce expressing each oncogene downstream of an leukemia in vivo. IRES in the MSCV vector that also contained Several recent studies have deployed modi- a green fluorescent protein (GFP) selectable fied retroviral transduction/transplantation or marker (MSCV-IRES-GFP [MIG]). In this transgenic models to examine Ras membrane study, transduced (GFP-positive) bone marrow trafficking, the roles of different effector path- HSPCs expressing oncogenic Nras caused mye- ways in leukemogenesis, the ability of oncogenic loid malignancies that resembled human chron- Ras expression to cooperate with other muta- ic myelomonocytic leukemia (CMML) at low tions to generate myeloid malignancies, and to viral titers and AML at higher titers. HSPCs perform preclinical testing of various therapeu- expressing oncogenic Kras uniformly caused a tic strategies. As described in detail in Philips CMML-like disease in recipient mice, the laten- (2017), Ras processing was initially pursued as cy of which correlated with viral titer. Finally, a therapeutic target in the 1980s and is an area expressing oncogenic Hras exclusively produced of renewed interest. Retroviral transduction of a

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A. Wandler and K. Shannon

palmitoylation-defective form of oncogenic ity of each fusion protein to activate a p53 re- Nras followed by transplantation of sorted sponse (Zuber et al. 2009). This transduction/ GFP-positive HSPCs abrogated myeloid disease transplantation model was used more recently and biochemical activation of downstream ef- in combination with a tetracycline-regulated fectors in recipient mice, suggesting a require- RNA interference (RNAi) system (Zuber et al. ment for Nras palmitoylation and subsequent 2011a) to identify BRD4 as a therapeutic target plasma membrane localization to induce mye- in AML (Zuber et al. 2011b), and to uncover loid malignancies (Cuiffo and Ren 2010). How- potential mechanisms of resistance to small ever, a subsequent study showed that expressing molecule inhibitors of this epigenetic modifier NrasG12D from the MSCV promoter has domi- (Rathert et al. 2015). nant negative effects on myeloid progenitor In summary, retroviral transduction/trans- colony growth and Ras-regulated activation of plantation and transgenic models have helped effector pathways ex vivo (Xu et al. 2012). These elucidate important aspects of Ras biology and data, in turn, raise the possibility that nonphy- have also been used to assess drug response and siologic levels of oncogenic N-RasG12D expres- resistance. As described below, the development sion impaired the growth of transduced HSPCs of “knockin” mouse strains has provided inves- before and after transplantation. Another recent tigators in the field with more robust and application of the MIG vector transduction/ reliable experimental models that overcome transplantation system assessed the ability of the potential confounding effects of nonphysio- “second-site” amino acid substitutions that logic levels of oncogenic Ras expression that impair the ability of K-RasG12D to bind Raf or also vary across different leukemias because of PI3 kinase (PI3K) to initiate leukemia in vivo differences in the genomic location of the inte- (Shieh et al. 2013). HSPCs transduced with grated transgene. these mutant KrasG12D oncogenes efficiently generated aggressive T-ALLs in recipient mice, and these leukemias restored downstream effec- IN VIVO EFFECTS OF NF1 INACTIVATION G12D/ tor activation by either acquiring “third-site” AND OF ENDOGENOUS KRAS NRASG12D EXPRESSION IN THE mutations within the Kras transgenes or by si- HEMATOPOIETIC COMPARTMENT lencing phosphatase and tensin homolog (PTEN) expression (Shieh et al. 2013). Induc- The World Health Organization classifies ible transgenic expression of oncogenic Nras JMML and CMML as MPN/MDS “overlap” under control of the Vav promoter caused disorders. These aggressive hematologic cancers mast cell leukemia that was reversed by admin- share overlapping clinical and biologic features, www.perspectivesinmedicine.org istering doxycycline to repress expression from a including excessive proliferation of cells in the tetracycline response element upstream of Nras. monocytic lineage, anemia, splenomegaly, pro- This inducible allele also cooperated with an gression to AML in a subset of patients, and Mll-Af9 fusion gene to produce AMLs that re- resistance to cytotoxic chemotherapy. One im- quired continuous oncogenic Nras expression portant difference between CMML and JMML to promote self-renewal of the leukemic popu- is that mutations in genes that broadly regulate lation (Kim et al. 2009; Sachs et al. 2014). Sim- epigenetic programs appear to initiate CMML, ilarly, retroviral transduction was used to coex- but not JMML (Jankowska et al. 2011; Sakagu- press oncogenic Nras with AML1/ETO or MLL chi et al. 2013; Stieglitz et al. 2015). This fusion genes under control of the MSCV pro- observation, in turn, suggests that fetal HSPCs moter in fetal liver cells, which were then trans- are uniquely susceptible to RAS-induced trans- planted into irradiated recipient mice to gener- formation in vivo. Indeed, JMML likely repre- ate AMLs. These leukemias were subsequently sents the clearest example of a human cancer used in preclinical studies to uncover differen- driven by aberrant Ras signaling as 90% of tial effects of standard induction chemotherapy JMML genomes harbor driver mutations in regimens resulting from a difference in the abil- one of five genes involved in Ras signaling

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Insights into Ras from Mouse Models of Hematologic Cancer

(NF1, NRAS, KRAS, PTPN11, CBL), but very pected finding was that endogenous Kras G12D few additional somatic alterations (Sakaguchi expression in primary hematopoietic cells re- et al. 2013; Stieglitz et al. 2015; reviewed in sulted in a modest increase in the levels of acti- Chang et al. 2014). vated Ras-GTP, but low basal ERK and Akt Given these human data, it was exciting phosphorylation (Braun et al. 2004). This ob- when investigators showed that expressing Cre servation provided one of the first hints that recombinase in the blood and bone marrow of hyperactive Ras signaling induces potent nega- mice harboring conditional mutant alleles of tive biochemical feedback in primary cells, Kras, Nras,orNf1 induced myeloid malignan- which was also observed in a subsequent anal- cies that recapitulated many aspects of JMML ysis of defined bone marrow cell populations and CMML. A key advance in the field was the isolated from Mx1-Cre; LSL-KrasG12D/þ mice development of a conditional Lox-Stop-Lox (Van Meter et al. 2007). (LSL) KrasG12D “knockin” allele that allowed To compare the effects of endogenous investigators to express oncogenic Kras from KrasG12D versus NrasG12D expression in colonic its endogenous locus following Cre-mediated epithelium, a conditional LSL-NrasG12D allele excision of the inhibitory LSL cassette (Jackson was expressed using the Fabpl-Cre promoter. et al. 2001). Although embryonic expression of Whereas mice expressing KrasG12D developed oncogenic Kras with ubiquitous or germline colonic hyperplasia, NrasG12D expression failed promoters was lethal, instilling an Adeno-Cre to induce proliferation of colon cells but in- vector into the lungs of LSL-KrasG12D mice gen- stead conferred resistance to apoptosis (Haigis erated multifocal adenocarcinomas that corre- et al. 2008). Similarly, inducing expression of lated with the viral titer (Jackson et al. 2001). To NrasG12D in the hematopoietic compartment examine the effects of oncogenic Kras ex- of Mx1-Cre; LSL-NrasG12D/þ mice had a differ- pression in the hematopoietic compartment, ent outcome than expressing KrasG12D in the two groups intercrossed LSL-KrasG12D and same strain background. Although Mx1-Cre; Mx1-Cre mice (Braun et al. 2004; Chan et al. LSL-KrasG12D/þ mice invariably died from pro- 2004). In this system, the interferon-inducible gressive MPNs by 4 months of age, Mx1-Cre; Mx1 promoter drives Cre recombinase expres- LSL-NrasG12D/þ mice exhibited indolent mye- sion following in vivo administration of polyI- loid disease that developed with greatly in- polyC (pI-pC), a synthetic double-stranded creased latency. Approximately 80% of these RNA that induces endogenous interferon ex- mice ultimately succumbed to a variety of he- pression (Ku¨hn et al. 1995). Mice expressing matologic malignancies by 1 year of age, which KrasG12D under control of the Mx1 promoter included MPNs, an MDS-like disorder, lympho- www.perspectivesinmedicine.org developed fully penetrant and aggressive proliferative disease, and histiocytic sarcoma. MPNs characterized by leukocytosis, splenome- Although bone marrow cells from Mx1-Cre; galy, and bone marrow myeloid hyperplasia. LSL-NrasG12D/þ mice showed cytokine-inde- Myeloid progenitor cells from these mice form pendent growth and GM-CSF hypersensitivity colonies in methylcellulose media in the ab- in methylcellulose cultures, these phenotypes sence of cytokine growth factors and also dis- were much less pronounced than in Kras mu- play hypersensitivity to granulocyte-macro- tant progenitors. Total Ras protein expression phage colony-stimulating factor (GM-CSF), a and Ras-GTP levels were also significantly high- cellular hallmark of JMML (Emanuel et al. er in myeloid lineage cells expressing KrasG12D 1991). Taken together, these studies established as compared to those expressing NrasG12D (Li a method for conditional expression of onco- et al. 2011). These differences in K-RasG12D and genic Ras alleles from their endogenous loci in N-RasG12D protein expression further suggested HSPCs, showed the ability of endogenous that Kras/Nras oncogene dosage might contrib- KrasG12D to perturb hematopoietic growth at ute to hematologic phenotypes. This hypothesis the single-cell level, and generated a new and was tested directly by generating Mx1-Cre mice robust model of JMML and CMML. An unex- expressing one or two NrasG12D alleles and

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showing that the homozygous NrasG12D/G12D mocytomas and JMML-like MPNs with incom- genotype caused an acute MPN that more close- plete penetrance (Jacks et al. 1994). These data ly resembled the aggressive myeloid disease are consistent with the tumor spectrum ob- induced by Kras G12D expression. In addition, served in persons with neurofibromatosis type transplanting bone marrow cells isolated from 1 (NF1), a familial cancer syndrome caused by heterozygous NrasG12D mutant mice into le- germline NF1 mutations. The tumor predispo- thally irradiated recipients resulted in predom- sitions and other phenotypic features of NF1 inantly CMML and rarely T-ALL, whereas disease, such as the role of germline and somatic transplantation of NrasG12D/G12D cells exclu- NF1 mutations in tumorigenesis, and the role of sively caused T-ALL (Wang et al. 2010). Analyz- NF1 GAP activity in regulating Ras signaling are ing hemizygous Mx1-Cre; NrasG12D/- mice discussed in detail in Cichowski (2017). Het- confirmed that gene dosage, and not tumor erozygous Nf1 inactivation also cooperated suppressor activity of wild-type (WT) Nras, with whole-body and focal radiation and the was responsible for the more aggressive biologic alkylating agent cyclophosphamide to induce behavior of homozygous mutant NrasG12D/G12D breast cancers, myeloid malignancies, sarcomas, hematopoietic cells (Xu et al. 2013). Finally, and pheochromocytomas (Chao et al. 2005; heterozygous NrasQ61L expression driven by Nakamura et al. 2011). Mx1-Cre caused an intermediate phenotype To circumvent the embryonic lethality re- of MPNs in mice that was more aggressive and sulting from homozygous Nf1 inactivation and penetrant than heterozygous NrasG12D expres- to create a mouse model of human NF1 disease, sion but less severe than homozygous NrasG12D a conditional mutant Nf1 allele was generated expression (Kong et al. 2016). Because N- (Zhu et al. 2001). Driving Cre recombinase ex- RasQ61L is more activated biochemically than pression from the Synapsin I promoter showed N-RasG12D, these studies provide additional ev- that tissue-restricted Nf1 inactivation caused idence that the degree and duration of oncogen- abnormal development of the cerebral cortex ic Ras output modulates cellular phenotypes. In and increased astrocyte proliferation (Zhu et this context, it is striking that NrasG12D, which al. 2001). Mx1-Cre; Nf1flox/flox mice developed has less potent effects on the proliferation and fully penetrant MPNs with a median survival of differentiation of primary hematopoietic cells 7.5 months—an intermediate hematologic phe- as compared to either NrasQ61L or Kras G12D,is notype between those observed in Mx1-Cre; also the most common mutation found in hu- LSL-NrasG12D/þ and Mx1-Cre; LSL-KrasG12D/ man blood cancers. Together, these data sup- þ mice. This myeloid disease models aspects of port the existence of substantial positive and JMML, including splenomegaly, bone marrow www.perspectivesinmedicine.org negative pressure that selects for the outgrowth infiltration by myeloid cells, and GM-CSF hy- of HSPCs with specific levels of aberrant Ras persensitivity. Transplanting Nf1-deficient bone pathway activation in vivo. marrow caused MPNs in lethally but not sub- The consequences of disrupting Nf1 were lethally irradiated recipient mice (Le et al. 2004). first examined using gene targeting to generate Mouse models exhibiting conditional Nf1 in- mice expressing a null allele (Brannan et al. activation in the hematopoietic compartment 1994; Jacks et al. 1994). Homozygous Nf1 mu- have facilitated comparing the consequences of tant embryos die in utero from cardiac defects. direct versus indirect activation of Ras signal- Interestingly, however, Nf1-deficient fetal liver ing, and have also provided an additional model cells displayed GM-CSF hypersensitivity in of MPNs for performing preclinical studies. methylcellulose cultures and caused JMML- like MPNs upon transplantation into lethally BIOLOGIC AND PRECLINICAL STUDIES irradiated recipients (Bollag et al. 1996; Largaes- IN KRAS, NRAS, AND NF1 MUTANT MICE pada et al. 1996). Heterozygous Nf1 mutant mice are phenotypically normal over the first In addition to generating models that recapitu- year of life and subsequently develop pheochro- late the characteristics of hematologic malig-

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Insights into Ras from Mouse Models of Hematologic Cancer

nancies driven by hyperactive Ras, conditional repopulating conditions (Sabnis et al. 2009). expression of KrasG12D or NrasG12D or ablation In contrast to Nf1 – / – and NrasG12D-expressing of Nf1 has elucidated fundamental aspects of HSPC, KrasG12D-expressing HSCs efficiently in- Ras biology and tumorigenesis. The relative fit- duced T-ALL under competitive repopulation ness of Nf1 mutant and WT HSPCs was assessed conditions. These aggressive cancers were char- by performing repopulation experiments (Fig. acterized by secondary mutations in Notch1, 2) in which defined numbers of sorted Nf1-de- demonstrating the acquisition of cooperating ficient bone marrow cells and WT “competi- mutations during progression to acute leuke- tors” were injected into irradiated recipient mia (Kindler et al. 2008; Sabnis et al. 2009). mice (Zhang et al. 2001). In these studies, Nf1 These studies show how genetically accurate mutant HSPCs outcompeted WT cells, but mouse models are robust platforms for identi- failed to induce MPNs unless a high ratio of fying cancer-initiating populations and for mutant-to-WT cells was injected (Zhang et al. addressing fundamental questions regarding 2001). Similarly, NrasG12D expression caused how endogenous oncogenic Ras expression increased proliferation of highly purified hema- perturbs stem cell fates. They also underscore topoietic stem cells (HSCs) and enhanced their the relative potency of oncogenic Kras versus competitive fitness but did not induce MPNs in oncogenic Nras or mutant Nf1 in leukemia recipients when coinjected with competitor initiation, albeit under the somewhat artificial cells. These data have important implications experimental condition of competitive repopu- for modeling leukemogenesis, as they indicate lation in irradiated congenic recipient mice. that WT cells can suppress the tumorigenic po- The observed GM-CSF hypersensitivity of tential of primary cells with oncogenic alter- Nf1-deficient progenitors in methylcellulose ations that activate Ras signaling. In addition, cultures raised the question of whether abnor- endogenous NrasG12D expression unexpectedly mal responses to exogenous stimuli are integral increased the frequency of cell divisions in a to the development of MPNs in vivo. Toaddress subpopulation of HSCs, but reduced this pa- this, fetal liver cells homozygous for mutations rameter in others. Enhanced self-renewal po- in both Nf1 and Gmcsf were transplanted into tential and competitive fitness was restricted WT or Gmcsf-deficient recipients (Birnbaum to the infrequently dividing HSC population et al. 2000). Remarkably, GM-CSF expression and was shown to be STAT5-dependent (Li in either donor cells or the host bone marrow et al. 2013). Oncogenic Kras expression microenvironment was sufficient to induce increased HSC proliferation and conferred a MPNs, but Gmcsf inactivation in both contexts competitive advantage over WT cells under severely prolonged disease latency. Further- www.perspectivesinmedicine.org

CD45.1+

Mix mutant HSCs Sort with WT “competitors” Harvest bone HSCs marrow

BoyJ (WT) 1.54DC

CD45.2+ Transplant into Analyze cells irradiated mice by FACS

CD45.2 Sort HSCs

B6 (mutant)

Figure 2. Competitive repopulation to assess fitness of hematopoietic stem cells (HSCs). Bone marrow cells are isolated from wild-type (WT) or mutant donor mice of different genetic backgrounds, then HSCs are sorted and mixed at a fixed ratio. These competitor cells are transplanted into irradiated recipient mice, then bone marrow is harvested and analyzed using fluorescence-activated cell sorting (FACS) to determine the percentage of mutant versus WT donor cells in the resulting population.

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more, transplanting doubly mutant bone mar- L744,832 inhibited H-Ras prenylation in recip- row cells from WT recipients with established ient mice but a substantial fraction of total Ras MPNs into secondary Gmcsf mutant recipients was processed normally (Mahgoub et al. 1999), caused disease regression, which was reversed which was almost certainly caused by “bypass” by exogenous GM-CSF administration (Birn- N-Ras and K-Ras prenylation by gerenylgerenyl baum et al. 2000). In a related study, a mutation transferase. This study accurately predicted the in the b common subunit of the GM-CSF re- disappointing efficacy of farnesyltransferase in- ceptor also attenuated the development of hibitors in the clinic. MPNs in Nf1 mutant mice (Kim et al. 2009). Directly inhibiting the oncogenic Ras/GAP These data indicate that Nf1 inactivation coop- switch continues to pose great challenges, and erates with upstream inputs from activated this has stimulated the development of numer- growth factor receptors to drive aberrant prolif- ous small molecule inhibitors of various Ras eration in vivo, and are consistent with bio- effectors. MEK is a particularly promising ther- chemical studies of primary Kras and Nf1 mu- apeutic target in many cancers, and the Food tant cells, showing that Ras effector pathways and Drug Administration has approved two are not fully activated under basal conditions allosteric inhibitors (trametinib and cobimeti- but respond robustly to growth factor stimula- nib) for advanced melanoma. Preclinical studies tion (Diaz-Flores et al. 2013). evaluating efficacy of the “first-generation” Two recent studies examined additional re- MEK inhibitor CI-1040 in Mx1-Cre; Nf1flox/flox quirements for the development of MPNs in mice with MPNs showed no beneficial effects Nf1 and Nras mutant mice. In the first, genetic (Lauchle et al. 2009). By contrast, Mx1-Cre; disruption of Erk1/2 abrogated myeloid disease LSL-KrasG12D mice that received PD0325901 in Mx1-Cre; Nf1flox/flox mice, supporting an es- (PD901), a “second-generation” MEK inhibit- sential role of aberrant Raf/MEK/ERK signal- or with optimized pharmacologic properties ing in induction of MPNs (Staser et al. 2013). (Brown et al. 2007), had remarkable hematolog- More recently, genetic ablation of Zdhhc9, ic improvement and greatly enhanced survival which encodes a palmitoyltransferase, impaired (Lyubynska et al. 2011). This study also uncov- the ability of NrasG12D to induce MPNs and T- ered an ability of PD901 to block GM-CSF hy- ALL (Liu et al. 2016). In addition to uncovering persensitivity in oncogenic Kras mutant bone genes required for the development of Ras-driv- marrow cells in vitro. However, Kras G12D hema- en diseases in hematopoietic cells, these studies topoietic cells persisted after treatment, indicat- credentialed the Raf/MEK/ERK pathway as ing that MEK inhibition rebalanced growth and a therapeutic target in MPNs characterized differentiation in vivo. Consistent with this www.perspectivesinmedicine.org by hyperactive Ras signaling and support inhi- observation, some Kras mutant mice progressed bition of the palmitoylation/depalmitoylation to T-ALL despite continuous treatment with cycle as a potentially effective therapeutic strat- PD901, which supports differential dependence egy for NRAS mutant hematologic cancers. on the Raf/MEK/ERK pathway in myeloid Nf1, Nras, and Kras mutant mice with versus lymphoid cells. To determine whether MPNs were also harnessed to directly test small the discordant results of these preclinical trials molecules with the potential to inhibit hyper- reflected differential sensitivity of Nf1 and Kras active Ras signaling. The initial preclinical trial mutant hematopoietic cells to MEK inhibition administered L744,832, a potent and selective or indicated that sustained Raf/MEK/ERK farnesyltransferase inhibitor (Kohl et al. 1995), pathway inhibition is essential for therapeutic to mice transplanted with homozygous Nf1-de- efficacy, PD901 was administered to Mx1-Cre; ficient fetal liver cells (Mahgoub et al. 1999). Nf1flox/flox mice with MPNs and induced pro- Although this compound reduced Raf/MEK/ found reductions in leukocytosis and spleno- ERK pathway activation and abrogated GM- megaly (Chang et al. 2013). As in mice with CSF colony growth ex vivo, it had no effect on Kras mutant MPNs, MEK inhibition failed to MPNs in vivo. Consistent with these data, eradicate Nf1 mutant HSPCs but rather exerted

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antiproliferative and prodifferentiation effects Normal Transient MPN JMML in the myeloid and erythroid lineages. Preclinical trials in mouse models of MPNs and other NF1-associated tumors (Jessen et al. 2013) informed a recent phase I clinical trial of Fetal HSC Fetal HSC Fetal HSC the MEK inhibitor selumetinib, which showed “Weak” “Strong” tumor regression in most children with plexi- germline germline mutation mutation form neurofibromas (Dombi et al. 2016). A national clinical trial of MEK inhibition in pa- tients with relapsed/refractory JMML is also expected to open later this year. While data Adult HSC Persistent Persistent from Kras and Nf1 mutant mice support the fetal HSC fetal HSC idea that MEK inhibition will induce hemato- logic improvement without eradicating mutant MEK inhibition HSPCs, certain clinical observations suggest that this approach has curative potential. Specifically, infants with developmental disor- ders of the Noonan syndrome (NS) spectrum Adult HSC (“cure”) sometimes develop a transient MPN that is clin- Potential therapeutic responses to MEK ically indistinguishable from JMML, but re- Figure 3. inhibition in juvenile myelomonocytic leukemia solves spontaneously (Kratz et al. 2005; Lauchle (JMML). Normal hematopoiesis is dynamically reg- et al. 2006; Loh 2011). The causative germline ulated during development with maturation from a PTPN11 and KRAS mutations in these patients fetal-like stem/progenitor to adult populations. The typically encode mutant proteins that are less observation of a self-limited JMML-like myeloprolif- activated biochemically than the strong somatic erative neoplasm (MPN) in infants with disorders of gain-of-function alleles detected in JMML the Noonan syndrome spectrum suggests that a threshold of aberrant Ras/Raf/MEK/ERK signaling (Keilhack et al. 2005; Schubbert et al. 2006, is required to fully transform fetal stem/progenitor 2007). In these cases, modestly activated SHP- cells. Based on this idea and preclinical data from Kras 2 and K-Ras mutant proteins likely fail to confer and Nf1 mutant mice with MPNs, possible beneficial a durable growth advantage. By contrast, stron- outcomes of sustained pharmacologic MEK inhibi- ger gain-of-function somatic mutations such as tion in JMML include (1) clinical improvement (re- K-RasG12D and SHP-2E76K appear to be capable duction in leukocyte counts, improvement of ane- of fully transforming fetal hematopoietic cells, mia, less splenomegaly) with persistence of the

www.perspectivesinmedicine.org malignant clone; or (2) eradication of the JMML leading to JMML. If this idea is correct, it raises clone caused by restoration of normal developmental the provocative possibility that pharmacologic processes regulating hematopoietic cell fates (dotted inhibition could reduce Raf/MEK/ERK signal- line). Clinical trials with molecular assessment of ing below a critical threshold level and thereby mutant allele burden in patient specimens are re- cure some JMML patients by allowing the nor- quired to investigate these potential mechanisms of mal developmental program to reassert itself response. HSC, Hematopoietic stem cell. (Fig. 3). The design of the forthcoming clinical trial includes correlative pharmacokinetic and molecular studies to directly address any under- reduced Raf/MEK/ERK pathway activation in lying mechanisms of response. response to GDC-0941, an observation that is Mx1-Cre; KrasG12D mice with MPNs were consistent with a previous study showing that also used to test the efficacy of GDC-0941, a PI3K functions both upstream and downstream pan-PI3K inhibitor (Akutagawa et al. 2016). of K-RasG12D in myeloid lineage cells stimulated Treatment resulted in disease remission similar with cytokine growth factors (Diaz-Flores et al. to that observed with PD901. However, bio- 2013). To more specifically inhibit PI3K/Akt chemical studies of bone marrow cells showed signaling, cohorts of Kras and Nf1 mutant

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mice were treated with the Akt inhibitor MK- ple independent Nf1 mutant AMLs, providing 2206 and preclinical efficacy was observed in evidence for cooperation between these two ge- both models (Akutagawa et al. 2016). These netic loci in myeloid leukemogenesis (Lauchle data highlight the utility of preclinical studies et al. 2009). in mouse models of oncongenic Ras-driven RIM was also performed in Mx1-Cre; MPNs to elucidate potential cross talk between NrasG12D/þ mice using a modified approach different Ras effector pathways. (Fig. 4B) in which mice were injected with MOL4070LTR shortly after birth and pI-pC was administered at weaning (Li et al. 2011). MODELS OF ACUTE LEUKEMIA This experimental system more closely recapit- CHARACTERIZED BY NF1 INACTIVATION ulates the pathogenesis of human AML in which OR ENDOGENOUS KRASG12D/NRASG12D RAS mutations generally comprise secondary EXPRESSION events that cooperate with antecedent oncogen- Whereas Nf1 inactivation and endogenous ic translocations or with mutations in genes KrasG12D or NrasG12D expression in hematopoi- encoding hematopoietic transcription factors etic cells generated robust and tractable models or proteins that broadly regulate epigenetic of CMML and JMML, these mice do not de- programs (Jan et al. 2012; Shlush et al. 2014; velop acute leukemia. This observation and Lindsley et al. 2015). The Nras mutant AMLs genomic analysis of human leukemias infer a generated in this study shared phenotypic fea- requirement of cooperating mutations for pro- tures with the M4/M5 subtype of human AML, gression to AML or ALL. Insertional mutagen- frequently exhibited loss of the WT Nras allele, esis is an unbiased forward genetic strategy for and showed heterogeneous activation of Ras ef- generating diverse primary leukemias. The first fector pathways. These AMLs were transplant- successful example of this method utilized the able into sublethally irradiated recipient mice, BXH-2 mouse strain that expresses a B-eco- and showed a clonal pattern of retroviral inser- tropic murine leukemia virus (MuLV) to induce tions that was stable upon secondary transplan- AML with long latency (Jenkins et al. 1982; tation. The most frequent common insertion Bedigian et al. 1984). Heterozygous Nf1 inacti- site was upstream of a gene encoding the zinc- vation both increased the incidence of myeloid finger transcription factor Evi-1 and resulted in leukemia and accelerated disease onset in BXH- a substantial increase in transcript expression 2 mice (Largaespada et al. 1996). Interestingly, specifically in leukemias harboring this integra- these Nf1 mutant AMLs frequently exhibited tion (Li et al. 2011). Interestingly, NRAS muta- loss of heterozygosity at the Nf1 locus, consis- tions in human AML are frequently associated www.perspectivesinmedicine.org tent with data from JMML patients with NF1 with both M4/M5 morphology and with EVI1 showing loss of the normal allele (Shannon translocations (Bowen et al. 2005; Bacher et al. et al. 1994). Over a decade later, retroviral inser- 2006). tional mutagenesis (RIM) was used to generate In contrast to Nf1 and Nras mutant mice, AMLs with unique cooperating genetic lesions Mx1-Cre; KrasG12D/þ mice that were injected in Mx1-Cre; Nf1flox/flox mice (Lauchle et al. with MOL4070LTR uniformly died from 2009). This experimental approach involved MPNs. However, transplanting their bone mar- simultaneously injecting newborn mice with row into sublethally irradiated recipients unex- pI-pC to induce Nf1 inactivation and with the pectedly generated T-ALLs in 80% and AMLs MOL4070LTR retrovirus to generate integra- in the remaining 20% of mice (Fig. 4C), which tions throughout the genome (Fig. 4A). In emerged with reduced latency compared to vi- this system, Nf1 cooperated with MOL4070LTR rus-injected WTanimals. Primary T-ALLs were to induce AMLs that were heterogeneous characterized by thymic masses, leukocytosis, with respect to morphology, myeloid surface extensive tissue infiltration, and an immature markers, and retroviral insertions. Myb was double-positive stage of lymphocyte develop- identified as a common insertion site in multi- ment. Secondary transplant recipients also

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Insights into Ras from Mouse Models of Hematologic Cancer

A Inject newborn mice with MOL4070LTR + pI-pC AML (75%)

flox/flox T-ALL (25%)

1 Retroviral mutagenesis fN ;erC- fN and Nf1 deletion ~150 days

1

xM

B Inject newborn mice Administer pI-pC at with MOL4070LTR weaning to express NrasG12D

G12D AML (100%) Outgrowth of Retroviral mutagenesis leukemic clone

21 days ~200 days

Mx1-Cre; LSL-Nras

C Inject newborn mice Administer pI-pC at Transplant bone marrow with MOL4070LTR weaning to express KrasG12D into irradiated recipients AML (20%)

G12D

s T-ALL (80%)

a r Outgrowth of

K-

LSL Retroviral mutagenesis Mice succumb to MPD leukemic clone

; 21 days 90 days 60–90 days

e

r

C

-1xM

Figure 4. Conditional Ras activation in the hematopoietic compartment. (A) Newborn mice are injected with MOL4070LTR to generate diverse genomic integrations and with polyI-polyC (pI-pC) to inactivate Nf1 in hematopoietic cells, resulting in predominantly acute myeloid leukemia (AML) with less frequent emergence of T-cell acute lymphoblastic leukemia (T-ALL). (B) Retroviral mutagenesis is performed in newborn mice, then pI-pC is administered at weaning to express NrasG12D in hematopoietic cells and generate AML. (C) Mice are injected with MOL4070LTR shortly after birth, then with pI-pC at weaning to activate KrasG12D. When these animals develop a lethal myeloproliferative disorder (MPD), bone marrow is transplanted into irradiated recipient mice to generate both AML and T-ALL.

developed T-ALL with more profound bone kemia. AMLs generated by transplanting Mx1- marrow infiltration and variable thymic in- Cre; KrasG12D/þ bone marrow into sublethally www.perspectivesinmedicine.org volvement (Dail et al. 2010). A common inser- irradiated recipients also contained clonal ret- tion site in Ikzf1, which encodes the lymphoid roviral integrations within genes such as Myb cell-specific transcription factor and known and Evi5 (Burgess et al. 2017). ALL tumor suppressor Ikaros (Rebollo and Taken together, data from RIM screens Schmitt 2003; Mullighan et al. 2007), was iden- performed with the same virus in mice express- tified in Kras-driven T-ALLs and integrations in ing mutant Nf1, Kras, and Nras in hematopoi- this gene appeared to result in proteins with etic cells highlight the extent to which specific dominant negative activity. Downstream Ras ef- Ras pathway alterations can modulate the spec- fector pathway activation was highly variable in trum of acute leukemias that develop. This find- Kras mutant and WT leukemias. Strikingly, so- ing is consistent with the variable latency and matic Notch1 mutations were identified in all penetrance of MPNs observed across the three Mx1-Cre; KrasG12D/þ T-ALLs examined (Dail genotypes described above. Importantly, these et al. 2010). This unanticipated observation panels of genetically diverse transplantable leu- demonstrates that cooperating events can arise kemias were subsequently deployed to perform from somatic alterations that are independent of controlled preclinical trials of signal transduc- retroviral insertions in RIM models of acute leu- tion inhibitors.

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PRECLINICAL TRIALS AND STUDIES rapid clearance of circulating leukemic blasts OF DRUG RESISTANCE IN MOUSE MODELS and a greater than threefold increase in median OF ACUTE LEUKEMIA survival compared to vehicle-treated animals. However, all of these mice eventually relapsed Acute leukemias generated by RIM recapitulate despite sustained treatment. AMLs that relapsed the genetic heterogeneity seen in advanced hu- after a prolonged response showed intrinsic man cancers, and transplantation into recipient drug resistance upon retransplantation and re- mice is an attractive preclinical platform (Fig. 5) treatment and also exhibited clonal evolution for assessing therapeutic responses and eluci- by the criterion of one or more novel retroviral dating mechanisms of drug resistance because integrations detected on Southern blots. Clon- (1) primary cancers are treated in immunocom- ing retroviral insertions from resistant AMLs petent mice; (2) retroviral integration patterns identified RasGrp1 overexpression and dis- can be used to track the emergence of drug re- ruption of one allele of Mapk14 as candidate sistant clones; and (3) relapsed leukemia cells resistance mechanisms that were validated func- can be retransplanted to verify intrinsic resis- tionally (Lauchle et al. 2009). tance, test alternative therapies, and validate Recipient mice transplanted with NrasG12D candidate resistance mechanisms. To examine AMLs showed a modest but significant survival the sensitivity of myeloid malignancies charac- benefit in response to treatment with PD901 or terized by Nf1 inactivation to MEK inhibition, trametinib. In contrast, these AMLs did not re- studies were initiated to assess the growth of spond to the pan-PI3K inhibitor GDC-0941 as a bone marrow cells from WT mice, Nf1 mutant single agent, and combining this drug with mice with MPNs, and Nf1-deficient AMLs gen- PD901 also had limited effects. As was observed erated by RIM in methylcellulose medium con- in Nf1-deficient AMLs, all of the recipient mice taining GM-CSF and various doses of CI-1040 given MEK and/or PI3K inhibitors relapsed on (Lauchle et al. 2009). In this in vitro system, treatment. However, none of these relapsed AML cells unexpectedly exhibited markedly en- AMLs harbored novel retroviral integrations hanced sensitivity to MEK inhibition compared and all showed a similar response to MEK inhi- to either WT cells or those harvested from mice bition upon retransplantation. Further analysis with MPNs. Transplanting Nf1-deficient AMLs revealed that MEK inhibition induces neither into recipient mice and treating them in vivo differentiation nor apoptosis of leukemia cells, revealed impressive efficacy of this compound but rather exerts its antileukemic effect by re- and of PD901, which was characterized by a ducing proliferation of NrasG12D AMLs (Bur- www.perspectivesinmedicine.org

Vehicle

Mutant genotype (Nf1-/-, NrasG12D, KrasG12D) Cryopreserved acute leukemia

Analyze response, clonal dynamics, and Drug resistance mechanisms

MOL4070LTR

Figure 5. Preclinical studies using acute leukemias generated by retroviral insertional mutagenesis (RIM). Mice expressing mutant Nf1, Nras,orKras under the control of Mx1-Cre are subjected to RIM to generate large panels of cryopreserved acute leukemias. These cells can be serially transplanted into recipient mice, exposed to various drugs or combinations in vivo, and then harvested at relapse and analyzed.

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Insights into Ras from Mouse Models of Hematologic Cancer

gess et al. 2014). Although this study showed mia, which exhibited remarkable clonal hetero- modest efficacy of potent small-molecule MEK geneity with numerous relapsed samples har- inhibitors, the lack of clonal evolution or ac- boring different novel retroviral integrations. quired resistance in relapsed AMLs was an im- T-ALLs that relapsed after a prolonged response portant limitation. Recently, an unbiased screen to treatment unexpectedly showed loss of retro- identified the creatine kinase pathway as a novel viral integrations and somatic mutations that therapeutic vulnerability in AML cell lines (Fe- activated Notch1 and a corresponding elevation nouille et al. 2017). This finding was validated in PI3K pathway activation (Dail et al. 2014). by transplanting primary Nras mutant AMLs These data have important implications for generated by RIM, treating them with a chem- the treatment of T-ALL, as they suggest that ical inhibitor of creatine biosynthesis, and the rational strategy of combining PI3K and showing that leukemia cells with an Evi1 inser- NOTCH1 inhibitors might drive clonal out- tion were much more sensitive to the drug (Fe- growth of drug-resistant cells. nouille et al. 2017). Treatment with PD901 also extended the CONCLUDING REMARKS survival of mice transplanted with KrasG12D AMLs with one leukemia showing an excep- The tractable nature of the hematopoietic com- tional response, harboring a novel integration partment and the availability of robust method- at relapse, and demonstrating both clonal evo- ologies for isolating and functionally analyzing lution and intrinsic drug resistance upon re- HSPCs in vivo have facilitated examining the transplantation/retreatment. Extensive analysis effects of Ras pathway activation on HSPC biol- of the parental AML and resistant subclone re- ogy and behavior. Extensive characterization of vealed a uniparental disomy event at the Kras mouse models of MPNs driven by mutant Nf1, locus that was followed by loss of the WT allele Kras,orNras has revealed important differences in the parental leukemia. This genetic configu- at the level of individual HSPCs and, more glob- ration conferred a competitive advantage upon ally, with respect to in vivo disease phenotypes these primary AML cells, but also rendered and the extent to which downstream Ras ef- them hypersensitive to MEK inhibition. Inter- fector pathways are activated following onco- estingly, the resistant AML exhibited trisomy gene expression. Taken together, these studies 6 and a Kras genotype of two mutant and one indicate that heterozygous Kras G12D is a more WT allele. These data showed a role for serial activating gain-of-function mutation than ho- genetic chances at the Kras locus in modulating mozygous Nf1 inactivation, which is, in turn, Raf/MEK/ERK pathway dependence, which more activating than a heterozygous NrasG12D www.perspectivesinmedicine.org was subsequently also observed in a panel of mutation. It will be interesting to perform sim- human colorectal cancer cell lines. Further- ilar comparisons in other tissue lineages. In ad- more, allelic imbalance at the KRAS locus was dition to demonstrating differential effects of identified in over half of a large panel of ad- individual mutant alleles, these studies also un- vanced human cancers with diverse histologies derscore the importance of posttranslational harboring oncogenic KRAS mutations (Burgess modifications of Ras in tumorigenesis. Con- et al. 2017). sistent with these findings, a recent genome- Preclinical studies in KrasG12D T-ALLs un- wide CRISPR-mediated screen to identify syn- covered a pattern of dependencies on the down- thetic lethal interactions found that genes stream Ras effector pathways that was distinct encoding proteins involved in Ras processing from AML. Whereas these leukemias showed a and components of the Raf/MEK/ERK path- modest response to PD901 and were refractory way were essential specifically in human AML to GDC-0941, treatment with both inhibitors cell lines harboring oncogenic RAS mutations markedly extended survival (Dail et al. 2010, (Wang et al. 2017). Another article in this col- 2014). As in the AML models, all recipient lection focuses on the topic of uncovering and mice ultimately died from drug-resistant leuke- validating candidate synthetic lethal interac-

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tions with oncogenic RAS (Aguirre and Hahn ACKNOWLEDGMENTS 2017). We are indebted to our long-term collaborators Developing predictive preclinical models Gideon Bollag, Kevin Haigis, Tyler Jacks, for testing new agents and drug combinations Michelle Le Beau, Scott Lowe, Luis Parada, remains a fundamental challenge for advancing David Tuveson, and Linda Wolff for reagents, cancer medicine. In this regard, the ability to advice, and assistance with much of the work transplant human and mouse leukemias into from our laboratory described in this review, cohorts of congenic recipient mice is a major which is supported by the National Institutes advantage. Collections of patient-derived xe- of Health (NIH) Grants R01 CA180037 and nografts (PDXs) are also valuable for preclini- R37 CA72614, by the Leukemia and Lymphoma cal evaluation because they are derived from Society Specialized Center of Research Grant human cancers and can reflect the heterogene- LLS 7019-04, by the Department of Defense ity of a given patient population. However, the (DOD) NF1 Research Program, and by the Rally use of immunocompromised mice for engraft- Foundation for Childhood Cancer Research. ment precludes examination of any potential A.W. is supported by a Postdoctoral Fellowship, effects of the microenvironment on response PF-14-070-01-TBG, from the American Cancer and resistance to therapeutic agents, and the Society, including a supplement from the Hill- reduced genetic complexity of PDX models crest Committee. K.S. is an American Cancer that results from the barrier of xenotransplan- Society Research Professor. tation (Klco et al. 2014) has potential limita- tions for characterizing relapse mechanisms. Whereas acute leukemias generated by RIM REFERENCES also recapitulate the genetic heterogeneity found in human tumors, they can be trans- Reference is also in this collection.

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A. Wandler and K. Shannon

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Mechanistic and Preclinical Insights from Mouse Models of Hematologic Cancer Characterized by Hyperactive Ras

Anica Wandler and Kevin Shannon

Cold Spring Harb Perspect Med published online August 4, 2017

Subject Collection Ras and Cancer in the 21st Century

Targeting Ras with Macromolecules MRAS: A Close but Understudied Member of the Dehua Pei, Kuangyu Chen and Hui Liao RAS Family Lucy C. Young and Pablo Rodriguez-Viciana Ras-Specific GTPase-Activating Proteins−− The Interdependent Activation of Structures, Mechanisms, and Interactions Son-of-Sevenless and Ras Klaus Scheffzek and Giridhar Shivalingaiah Pradeep Bandaru, Yasushi Kondo and John Kuriyan Ras-Mediated Activation of the Raf Family Targeting the MAPK Pathway in RAS Mutant Kinases Cancers Elizabeth M. Terrell and Deborah K. Morrison Sarah G. Hymowitz and Shiva Malek Posttranslational Modifications of RAS Proteins Ras and the Plasma Membrane: A Complicated Ian Ahearn, Mo Zhou and Mark R. Philips Relationship Yong Zhou, Priyanka Prakash, Alemayehu A. Gorfe, et al. Kras in Organoids Kras and Tumor Immunity: Friend or Foe? Derek Cheng and David Tuveson Jane Cullis, Shipra Das and Dafna Bar-Sagi KRAS: The Critical Driver and Therapeutic Target Synthetic Lethal Vulnerabilities in KRAS-Mutant for Pancreatic Cancer Cancers Andrew M. Waters and Channing J. Der Andrew J. Aguirre and William C. Hahn The K-Ras, N-Ras, and H-Ras Isoforms: Unique Efforts to Develop KRAS Inhibitors Conformational Preferences and Implications for Matthew Holderfield Targeting Oncogenic Mutants Jillian A. Parker and Carla Mattos PI3K: A Crucial Piece in the RAS Signaling Puzzle Genetically Engineered Mouse Models of Agata Adelajda Krygowska and Esther Castellano K-Ras-Driven Lung and Pancreatic Tumors: Validation of Therapeutic Targets Matthias Drosten, Carmen Guerra and Mariano Barbacid

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