Search

Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2), associates with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore Srsf2P95H interaction with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFβ1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wildtype animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.

Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3′ end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3′ splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias. Loss of function of the minor spliceosome component ZRSR2 enhances hematopoietic stem cell self-renewal through minor intron retention of its target LZTR1, which is a regulator of RAS-related GTPases. Minor intron retention of LZTR1 was also identified in Noonan syndrome and diverse solid tumor types.

BACKGROUND: Covalent (irreversible) Bruton’s tyrosine kinase (BTK) inhibitors have transformed the treatment of multiple B-cell cancers, especially chronic lymphocytic leukemia (CLL). However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Noncovalent (reversible) BTK inhibitors overcome this mechanism and other sources of resistance, but the mechanisms of resistance to these therapies are currently not well understood. METHODS: We performed genomic analyses of pretreatment specimens as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the noncovalent BTK inhibitor pirtobrutinib. Structural modeling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to noncovalent BTK inhibitors. RESULTS: Among 55 treated patients, we identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. We found mutations (V416L, A428D, M437R, T474I, and L528W) that were clustered in the kinase domain of BTK and that conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or phospholipase C gamma 2 (PLCγ2), a signaling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell–receptor signaling persisted despite continued therapy with noncovalent BTK inhibitors. CONCLUSIONS: Resistance to noncovalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. These data suggested new mechanisms of genomic escape from established covalent and novel noncovalent BTK inhibitors. (Funded by the American Society of Hematology and others.)

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Significance Myelodysplastic syndromes (MDS) are the most commonly diagnosed malignancy of the elderly in the United States, but what causes severe anemia in MDS has been unknown. Our findings provide a detailed mechanism underlying the origins of severe anemia and other cardinal phenotypes in MDS patients harboring SF3B1 mutations, which are found in about a quarter of all MDS patients. In addition, we define a role of MAP3K7 and a MAP3K7-p38 MAPK axis in human terminal erythroid differentiation and created a SF3B1 cell model that recapitulates many of the terminal erythroid differentiation events of mutant SF3B1 MDS patients, which should prove valuable for developing novel drugs and therapies., SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

SF3B1 is the most commonly mutated RNA splicing factor in cancer1–4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L5–7. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies. A range of SF3B1 mutations promote tumorigenesis through the repression of BRD9, a core component of the non-canonical BAF complex, and correcting BRD9 mis-splicing in these SF3B1-mutant cells suppresses tumour growth.

SF3B1 is the most frequently mutated RNA splicing factor in multiple neoplasms, including ~25% of myelodysplastic syndromes (MDS) patients. Mortality in MDS frequently results from severe anemia, but the underlying mechanism is largely unknown. Here we elucidate the detailed, elusive pathway by which SF3B1 mutations cause anemia. We demonstrate, in CRISPR-edited cell models, normal human primary cells, and MDS patient cells, that mutant SF3B1 induces a splicing error in transcripts encoding the kinase MAP3K7, resulting in reduced MAP3K7 protein levels and deactivation of downstream target p38 MAPK. We show that disruption of this MAP3K7-p38 MAPK pathway leads to premature downregulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation and apoptosis. As a result, the overproduced, late staged erythroblasts undergo apoptosis and are unable to mature in the bone marrow. Our findings provide a detailed mechanism explaining the origins of anemia in MDS patients harboring SF3B1 mutations.

The purpose of this study is to launch a novel biomarker-based targeted therapy that may result in improved precision and efficacy for the treatment of pancreatic ductal adenocarcinoma (PDAC). We discovered that Keratin 17 (K17), an oncofetal intermediate filament and nuclear oncoprotein expressed in 50% of PDAC cases, is a biomarker of the most aggressive and treatment-resistant form of PDAC. We set out to validate K17 as a druggable target and to identify K17-targeting mechanisms. In pre-clinical mouse models, animals bearing K17+ PDACs display the shortest survival, suggesting that K17 drives tumor aggression and should be explored as a potential therapeutic target. We used the following two approaches aimed to inhibit K17 nuclear shuttle functions, in order to identify therapeutic strategies: 1) Targeting K17 functional domains: By protein-sequence modeling analyses and site-directed mutagenesis, we identified functional domains in K17 (K17-FDs) that bind to, shift the subcellular localization of, and promote the degradation of nuclear tumor suppressors (e.g. p27). Using SILAC mass spectrometry of nuclear proteomes from isogenic human cells, we found that 80% of the nuclear proteome is altered by K17, that half of these proteins encode the domain targeted by the K17-FDs, and that a large proportion of these proteins are involved in gene expression. Animals harboring tumors with mutated K17-FDs lived two-times longer than control animals bearing K17+ PDACs, suggesting that the K17-FDs may be therapeutic targets. We are currently testing small molecule and peptide inhibitors to target the K17-FDs in vitro and in vivo, as a discovery approach to design small-molecule inhibitors of K17. 2) Targeting K17 nuclear export: K17 impacts the nuclear export of ~50% of the proteome and depends on exportin-1 (XPO1). We found that K17+ PDAC cells were more sensitive to Selective Inhibitor of Nuclear Export (SINE) therapy, leading to a 2-fold increase in cell death compared to control cells, suggesting that nuclear export by K17 in PDAC cells could be therapeutically targeted. SINE therapy is currently FDA-approved for treatment of other cancer types and repurposing for the treatment of K17-positive PDACs may result in enhanced therapeutic efficacy. In conclusion, these studies provide the basis for the development of novel biomarker-based therapeutic approaches using small-molecule inhibitors, to target K17 active sites or the pathways altered by this protein in PDAC. Citation Format: Cindy V. Leiton, Chun-Hao Pan, Ji Dong Bai, Lucia Roa-Peña, Sruthi Babu, Alex Penson, Nashaat Turkman, Richard Moffitt, Markus Seeliger, David Talmage, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Therapeutic targeting of keratin 17 functional domains and nuclear export uncover therapeutic vulnerabilities of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-040.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

Dissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually

The purpose of this study is to launch a novel biomarker-based targeted therapy that may result in improved precision and personalized medicine for pancreatic ductal adenocarcinoma (PDAC). We discovered that keratin 17 (K17), an intermediate filament (IF), is a nuclear oncoprotein found in 50% of PDAC cases, identifying the most aggressive and treatment-resistant form of PDAC and functioning as a biomarker. We set out to validate K17 as a druggable target and to identify K17-targeting mechanisms. In preclinical mouse models, animals bearing K17-expressing PDACs display the shortest survival interval, suggesting that K17 drives tumor aggression and is a potential therapeutic target. We used the following two approaches aimed to inhibit K17 nuclear shuttle functions in order to identify potential therapeutic strategies. 1) Targeting K17 functional domains: By protein-sequence modeling analyses and site-directed mutagenesis, we identified functional domains in K17 (K17-FDs) that bind to, shift the subcellular localization of, and promote the degradation of nuclear tumor suppressors (i.e., p27). Using SILAC mass spectrometry analysis of nuclear proteomes from isogenic human cells, we found that 80% of the nuclear proteome is altered by K17, that half of these proteins encode the domain targeted by the K17-FDs, and that a large proportion of these proteins are involved in gene expression. Animals harboring tumors with mutated K17-FDs lived two times longer than control animals, suggesting that the K17-FDs may be therapeutic targets. We are currently testing small-molecule and peptide inhibitors to target the K17-FDs in vitro and in vivo, as a discovery approach to design small-molecule inhibitors of K17. 2) Targeting K17 nuclear export: K17 impacts the export of ~50% of the proteome and depends on exportin-1 (XPO1) for nuclear export. We found that PDAC cells expressing K17 were more sensitive to Selective Inhibitor of Nuclear Export (SINE) therapy, leading to a 2-fold increase in cell death compared to control cells, suggesting that nuclear export is a necessary oncogenic pathway exploited by K17 in PDAC cells. SINE therapy is currently FDA approved for treatment of other cancer types, and repurposing for the treatment of K17-positive PDACs may result in enhanced therapeutic efficacy. In conclusion, these studies provide the basis for novel biomarker-based therapeutic approaches for the most aggressive PDAC cases using small-molecule inhibitors that directly target K17 active sites or the pathways altered by this protein. Citation Format: Cindy V. Leiton, Chun-Hao Pan, Ji Dong Bai, Lucia Roa-Peña, Sruthi Babu, Alex Penson, Nashaat Turkman, Richard Moffitt, Markus Seeliger, David Talmage, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Therapeutic targeting of keratin 17 and nuclear export uncover therapeutic vulnerabilities of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C32.

Human cancers arise from environmental, heritable, and somatic factors. While each has been studied extensively, how these factors interact to drive tumorigenesis and disease phenotypes is poorly understood. Here, we integrated germline pathogenicity with somatic alterations inferred from the prospective sequencing of 17,152 advanced cancer patients across 55 cancer types to explore their interplay in tumorigenesis. Leveraging expert curated germline variant calls from 5,358 patients, we developed a combined machine learning and risk stratification-based approach for pathogenicity assessment. Overall, 10.3% of patients harbored a pathogenic allele with penetrance levels ranging from high to low, and an additional 6.3% with a variant of uncertain penetrance. Integrating high-precision zygosity inference in the concomitant cancer diagnoses, we found that only carriers of high penetrance germline pathogenic alleles that are biallelically inactivated in their tumors had an earlier age of onset (51y in carriers vs. 58y in germline WT, p-value=3.5e-12), and an elevated rate of multiple independent cancer diagnoses (23% in carriers vs 13% in germline WT, p-value=1.6e-7). Broadly, penetrance dictated somatic dependence, with only those higher penetrant germline pathogenic alleles associated with lineage-dependent selective pressure for somatic biallelic inactivation (p-value Citation Format: Chaitanya Bandlamudi, Preethi Srinivasan, Philip Jonsson, Yelena M. Kemel, Shweta S. Chavan, Allison L. Richards, Alex Penson, Craig M. Bielski, Chris Fong, Aijazuddin Syed, Gowtham Jayakumaran, Meera Prasad, Jason Hwee, Nikolaus Schultz, Semanti Mukherjee, Vijai Joseph, Diana Mandelkar, Ozge Birsoy, Liying Zhang, Jinru Shia, Ahmet Zehir, Marc Ladanyi, David M. Hyman, Kenneth Offit, Mark E. Robson, David B. Solit, Zsofia K. Stadler, Michael F. Berger, Barry S. Taylor. Zygosity, lineage, and penetrance dictate the role of germline pathogenicity in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 729.

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (

Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children. Altogether 90% of pediatric ALL patients achieve a complete hematologic remission with current high dose combination chemotherapy and 80% of them remain leukemia free. However, the outcome for patients showing refractory disease or those whose leukemia relapses after an initial transient response remains disappointingly poor with cure rates of less than 40%. To investigate genetic drivers of relapse and resistance and explore the specific roles of clonal evolution in disease progression and relapse here we performed whole-exome sequence analysis of matched diagnosis, germline (remission) and relapse DNA samples in a panel of 55 pediatric ALL patients including 33 T-cell ALLs and 22 B-cell precursor ALLs. These analyses identified an average of 9 mutations present in diagnostic samples and 17 mutations in relapsed leukemia DNAs. Phylogenetic tree analysis for each of the 48 cases with optimal variant call parameters analyzing their clonal evolution dynamics during disease progression, combined with whole genome sequencing of targeted samples with low exonic mutation input, showed that branched evolution in which relapse clones contain some, but not all genetic lesions present in the major clone at diagnosis as the primary mechanism driving tumor progression and relapse present in 45/48 (94%) cases. In addition, and consistent with previous reports we identified the presence of chemotherapy associated mutations in NT5C2 (10/55), TP53 (3/55), CREBBP (4/55) and the NR3C1 glucocorticoid receptor gene (2/55). However, and most strikingly, 23/27 (85%) recurrently mutated genes in this series with mutations preferentially selected or retained at the time of relapse (mutation never lost in the relapse clone) were not implicated in relapse ALL before (HTR3A, MED12, USP9X, CACNA1H, ODZ3, AACS, SAMD4A, ANO5, PAPPA, NAALADL2, HIST3H2A, FZD7, TBX15, NEB, GREB1L, PLXNA4, SGK223, TSC1, PTPRG, FGF10, SYCP2, TRPM3 and EYS). A branched pattern of genetic evolution and the presence of recurrent mutations selected at relapse support that chemotherapy imposes a strong Darwinian genetic selection in leukemic cell populations. In this context it is worth noting that RAS-MAPK pathway activating mutations in NRAS, KRAS and PTPN11 were present in 24/55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, while in others, RAS mutant clones present at diagnosis were replaced by RAS wild type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Most notably, and in agreement with this hypothesis, inducible expression of mutant KRAS in human ALL lines demonstrate that oncogenic KRAS G12D induces methotrexate resistance, but also improves leukemia response to vincristine; a phenotype perfectly recapitulated in a isogenic ALL leukemia model generated from a conditional inducible Kras G12D knockin mice. Mechanistically, KRAS G12 expression induces MAPK dependent abrogation of methotrexate induced apoptosis. Moreover, Kras mutant tumors show enhanced G2/M cell cycle arrest and apoptosis upon spindle poisoning with vincristine, a phenotype linked with increased PLK phosphorylation and transcriptional down-regulation of mitotic genes. Finally clonal competition assays demonstrate that the differential response to methotrexate and vincristine in isogenic Kras wild type and Kras mutant ALL cells results in clonal dominance of Kras G12D populations in cultures treated with methotrexate, while Kras wild type cells are selected the context of vincristine treatment. In all these results show novel insight on the genetics and mechanisms of clonal selection, disease progression and relapse in ALL and demonstrate a previously unrecognized dual role of RAS mutations in chemotherapy response. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.