Oncogenic NRASG12D Research Articles

Abstract B11: Mouse models of melanoma driven by oncogenic NRAS

Abstract Melanoma is the most deadly form of skin cancer and is a disease that is rising in incidence. Approximately 20% of all melanomas harbor activating mutations in NRAS. We previous used inducible-conditional approaches to express oncogenic NRAS (NRASG12D) at physiological levels in mouse melanocytes. When expressed in adult mouse melanocytes, NRASG12D induced skin darkening and blue nevus-like lesions but did not induce cutaneous melanoma. When expressed during embryogenesis, NRASG12D induced congenital nevi and leptomeningeal melanocytosis that replicated the cardinal features of human neurocutaneous melanosis. To extend these studies, we have examined how ERK signal strength affects NRASG12D-driven melanomagenesis. We previously demonstrated that kinase-dead BRAF drives paradoxical hyper-activation of ERK downstream of oncogenic RAS, so to investigate how ERK signal strength affects RAS-driven melanomagenesis, we expressed NRASG12D and kinase-dead BRAF (BRAFD594A) in adult mouse melanocytes. BRAFD594A did not induce any skin lesions. In accordance with our previous study, NRASG12D induced paucicellular nevi composed of pigmented dendritic melanocytes in the papillary and superficial reticular dermis. These lesions resembled nevus of Ota, but none of the mice developed melanoma. When BRAFD594A and NRASG12D were co-expressed, we observed the paucicellular nevi seen in NRASG12D mice, but additionally, we observed a proliferation of nevi in the deep reticular dermis, the hypodermis and enveloping the cutaneous adnexae. The cells in these lesions were heavily pigmented epitheliod or ovoid melanocytes and the lesions were reminiscent of dermal melanocytosis with scattered foci of blue nevi. More importantly, these mice developed melanoma that presented with two distinct histological types. The majority of the tumours presented as hypo-pigmented lesions that extended from the upper dermis into the subcutaneous layers, leaving a narrow or absent grenz zone and frequent focal ulceration. They were amelanotic and the predominant cells were undifferentiated atypical spindle melanocytes. The tumours replaced the normal architecture of the upper dermis, invading the adjacent and subcutaneous structures, and occasionally presented nevi remnants at their poorly defined borders. Less common, the tumours were hyper-pigmented lesions that arose in the deep reticular dermis, leaving a wide grenz zone and only occasionally ulcerating the epidermis. The tumour cells were predominantly heavily pigmented, pleomorphic epithelioid melanocytes, abutting into the deeper layers of the subcutis and the lesions resembled animal-type melanoma in humans. Thus, we show that ERK signal strength is critical for NRAS-driven melanomagenesis and this pathway drives different types of lesion that resemble human nevi and melanoma. Citation Format: Malin Pedersen, Amaya Viros, Richard Marais. Mouse models of melanoma driven by oncogenic NRAS. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B11. doi: 10.1158/1557-3125.RASONC14-B11

Abstract SY13-04: Stem cell self-renewal and cancer

Abstract Haematopoietic stem cell self-renewal and leukemogenesis “Pre-leukemic” mutations are thought to promote clonal expansion of haematopoietic stem cells (HSCs) by increasing self-renewal and competitiveness; however, mutations that increase HSC proliferation tend to reduce competitiveness and self-renewal potential, raising the question of how a mutant HSC can sustainably outcompete wild-type HSCs. Activating mutations in NRAS are prevalent in human myeloproliferative neoplasms and leukemia. We found that a single allele of oncogenic NrasG12D increases HSC proliferation but also increases reconstituting and self-renewal potential upon serial transplantation in irradiated mice, all prior to leukemia initiation. NrasG12D also confers long-term self-renewal potential upon multipotent progenitors. To explore the mechanism by which NrasG12D promotes HSC proliferation and self-renewal we assessed cell cycle kinetics using H2B-GFP label retention and BrdU incorporation. NrasG12D had a bimodal effect on HSCs, increasing the rate at which some HSCs divide and reducing the rate at which others divide. This mirrored bimodal effects on reconstituting potential as rarely dividing NrasG12D HSCs outcompeted wild-type HSCs while frequently dividing NrasG12D HSCs did not. NrasG12D had these effects by promoting STAT5 signaling, inducing different transcriptional responses in different subsets of HSCs. One signal can therefore increase HSC proliferation, competitiveness, and self-renewal through bimodal effects on HSC gene expression, cycling, and reconstituting potential. We have also developed methods for studying protein synthesis by individual stem cells in vivo. Currently, there are almost no data on protein synthesis in any somatic stem cell. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosomal function in Rpl24Bst/+ mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24Bst/+ cell-autonomously rescued the effects of Pten deletion in HSCs, blocking the increase in protein synthesis, restoring HSC function, and delaying leukemogenesis. Pten deficiency thus depletes HSCs and promotes leukemia partly by increasing protein synthesis. Increased or decreased protein synthesis impairs HSC function. Citation Format: Sean J. Morrison. Stem cell self-renewal and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY13-04. doi:10.1158/1538-7445.AM2014-SY13-04

NrasG12D oncoprotein inhibits apoptosis of preleukemic cells expressing Cbfβ-SMMHC via activation of MEK/ERK axis

AbstractAcute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFβ-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFβ-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used NrasLSL-G12D; Cbfb56M knock-in mice to show that allelic expression of oncogenic NrasG12D and Cbfβ-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. NrasG12D and Cbfβ-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfβ-SMMHC. Furthermore, NrasLSL-G12D; Cbfb56M leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfβ-SMMHC and NrasG12D promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inversion(16) AML–targeted therapies.

The role of RAS effectors in BCR/ABL induced chronic myelogenous leukemia

BCR/ABL is the causative agent of chronic myelogenous leukemia (CML). Through structure/function analysis, several protein motifs have been determined to be important for the development of leukemogenesis. Tyrosine177 of BCR is a Grb2 binding site required for BCR/ABL-induced CML in mice. In the current study, we use a mouse bone marrow transduction/transplantation system to demonstrate that addition of oncogenic NRAS (NRASG12D) to a vector containing a BCR/ABL(Y177F) mutant "rescues" the CML phenotype rapidly and efficiently. To further narrow down the pathways downstream of RAS that are responsible for this rescue effect, we utilize well-characterized RAS effector loop mutants and determine that the RAL pathway is important for rapid induction of CML. Inhibition of this pathway by a dominant negative RAL is capable of delaying disease progression. Results from the present study support the notion of RAL inhibition as a potential therapy for BCR/ABL-induced CML.

Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase

Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation.Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro.We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma.Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells. Disclosures:No relevant conflicts of interest to declare.

Dominant Role of Oncogene Dosage and Absence of Tumor Suppressor Activity in Nras-Driven Hematopoietic Transformation

Biochemical properties of Ras oncoproteins and their transforming ability strongly support a dominant mechanism of action in tumorigenesis. However, genetic studies unexpectedly suggested that wild-type (WT) Ras exerts tumor suppressor activity. Expressing oncogenic Nras(G12D) in the hematopoietic compartment of mice induces an aggressive myeloproliferative neoplasm that is exacerbated in homozygous mutant animals. Here, we show that increased Nras(G12D) gene dosage, but not inactivation of WT Nras, underlies the aggressive in vivo behavior of Nras(G12D/G12D) hematopoietic cells. Modulating Nras(G12D) dosage had discrete effects on myeloid progenitor growth, signal transduction, and sensitivity to MAP-ERK kinase (MEK) inhibition. Furthermore, enforced WT N-Ras expression neither suppressed the growth of Nras-mutant cells nor inhibited myeloid transformation by exogenous Nras(G12D). Importantly, NRAS expression increased in human cancer cell lines with NRAS mutations. These data have therapeutic implications and support reconsidering the proposed tumor suppressor activity of WT Ras in other cancers. Understanding the mechanisms of Ras -induced transformation and adaptive cellular responses is fundamental. The observation that oncogenic Nras lacks tumor suppressor activity, whereas increased dosage strongly modulates cell growth and alters sensitivity to MEK inhibition, suggests new therapeutic opportunities in cancer.

Primary Melanoma of the CNS in Children Is Driven by Congenital Expression of Oncogenic NRAS in Melanocytes

NRAS mutations are common in human melanoma. To produce a mouse model of NRAS-driven melanoma, we expressed oncogenic NRAS (NRAS(G12D)) in mouse melanocytes. When NRAS(G12D) was expressed in the melanocytes of developing embryos, it induced melanocyte proliferation and congenital melanocytic lesions reminiscent of human blue nevi but did not induce cutaneous melanoma. Unexpectedly, however, it did induce early-onset primary melanoma of the central nervous system (CNS). The tumors were rapidly proliferating and caused neurologic symptoms, rapid health deterioration, and death. NRAS is not a common driver oncogene of primary melanoma of the CNS in adults, but we report two cases of primary melanoma of the CNS in children, both of which carried oncogenic mutations in NRAS. We conclude that acquisition of somatic mutations in NRAS in CNS melanocytes is a predisposing risk factor for primary melanoma of the CNS in children, and we present a mouse model of this disease. We show that the acquisition of NRAS mutations in melanocytes during embryogenesis is a risk factor for early-onset melanoma of the CNS. We have developed a powerful mouse model to study this rare but devastating childhood disease, and to develop therapeutic approaches for its treatment.

Targeting BCL6-Mediated Drug-Resistance in High-Risk Childhood ALL

Abstract 776 Background:BCL6 is known as a protooncogene and transcriptional repressor in Diffuse Large B cell lymphoma (DLBCL), where BCL6 is frequently involved in chromosomal rearrangements. We recently found that BCL6 mediates a novel form of drug resistance to tyrosine kinase inhibitors (TKI) in Ph+ ALL and CML (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). This was based on the finding that Ph+ ALL and CML cells respond to TKI-mediated inhibition of BCR-ABL1 kinase activity by upregulation of BCL6, which protects from p53-mediated apoptosis. Our current study was prompted by the finding that high expression levels of BCL6 represent a predictor of poor clinical outcome in children with Non-Ph ALL. We therefore performed detailed studies of expression and function of BCL6 in various subtypes of Non-Ph childhood ALL. Results:In a gene expression analysis of 207 children with high-risk B cell precursor ALL (COG P9906 trial), we found that high expression levels of BCL6 at the time of diagnosis represents a strong predictor of poor overall (OS; p=0.007) and relapse-free (RFS; p=0.02) survival. Also, high expression levels of BCL6 are predictive of positive minimal residual disease (MRD+) status on day 29 after the onset of chemotherapy. For 49 patients in this clinical trial, matched sample pairs at diagnosis and relapse were available. In these patients, BCL6 expression was significantly higher at relapse compared to diagnosis (p=0.003). We next studied BCL6 mRNA (n=122) and protein (n=21) levels in a larger cohort of primary childhood ALL patient samples and found that BCL6 expression levels are particularly high in MLL-AF4 infant ALL. This finding was unexpected and we tested if the MLL-AF4 oncogene drives aberrant BCL6 expression in these cells. First, ChIP-analysis revealed that the chimeric oncoprotein MLL-AF4 directly binds to the BCL6 promoter, suggesting that MLL-AF4 may indeed drive BCL6 expression. In support of this hypothesis, BCL6 Western blot analyses of inducible MLL-AF4-transgenic pro-B cells demonstrated that activation of the MLL-AF4 transgene is sufficient to induce ∼10-fold upregulation of BCL6 protein levels. We conclude that aberrant expression of BCL6 in childhood ALL can be the direct consequence of MLL-AF4 activity. To investigate the potential function of BCL6, we used a genetic mouse model of childhood ALL based on bone marrow precursor cells from BCL6−/− mice. Since mutations in the RAS pathway are found in about 30% of childhood ALL cases, we transformed B cell progenitor cells from BCL6−/− and wildtype mice with oncogenic NRASG12D, which represents the most frequent RAS lesion in B cell lineage leukemia. Surprisingly, BCL6-deficiency results in a failure of NRASG12D ALL cells to initiate leukemia, while NOD/SCID mice that were transplanted with BCL6 wildtype NRASG12DALL succumbed to the disease. Clinical relevance:To verify if these observations are relevant to human disease, we transduced primary human childhood ALL xenografts with MLL-AF4 gene rearrangement with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if BCL6 inhibition is of potential use for children with MLL-AF4 leukemia, we used a recently developed retro-inverso BCL6 peptide inhibitor (RI-BPI, Cerchietti et al., Blood 2009). RI-BPI is able to inhibit BCL6 function and is currently under clinical development for the treatment of BCL6-dependent DLBCL. Treatment of the primary human MLL-AF4 ALL xenograft cells with RI-BPI compromised colony formation in methylcellulose and leukemia-initiation in transplant recipient mice and had a strong synergistic effect when combined with Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. Conclusions:These findings identify BCL6 as a therapeutic target in high-risk childhood leukemia and its pharmacological inhibition as a novel strategy as therapeutic adjuvant. Interestingly, MLL-AF4 ALL cells exhibit constitutively high expression levels of BCL6. Aberrant expression of BCL6 in MLL-AF4 ALL is the direct consequence of MLL-AF4 activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors (e.g. RI-BPI) with standard chemotherapy as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures:No relevant conflicts of interest to declare.

Oncogenic Nras Increases Hematopoietic Stem Cell Proliferation and Self-Renewal Through a Bimodal Effect

AbstractAbstract 119Accumulating evidence suggests that most leukemias are initiated by rare leukemic stem cells (LSC) that are transformed from the normal hematopoietic stem cells and progenitors (HSC/P) by genetic lesions that lead to activation of oncogenes and inactivation of tumor suppressor genes. However, the signaling mechanisms by which these genes transform HSC/P into LSC are poorly understood. Activating mutations of NRAS and KRAS are highly prevalent in acute myeloid leukemia (AML), some myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). In addition other leukemia associated genetic lesions, such as the BCR-ABL fusion, PTPN11 mutations, FLT3 internal tandem duplications, and NF1 inactivation all deregulate Ras signaling. We previously developed a mouse strain that conditionally expresses an oncogenic NrasG12D allele from the endogenous locus. This consistently resulted in an indolent MPD with delayed onset and prolonged survival in Mx1-cre, NrasG12D/+ mice (referred to as NrasG12D). Oncogenic NrasG12D, however, cooperated with the MOL4070LTR retrovirus to induce AMLs that share molecular and morphologic features with human M4/M5 AML. Here we report that NrasG12D directly affects HSC/P functions. While normal HSCs must remain quiescent to maintain the long term self-renewal capacity and mutations that drive HSC into cycle often lead to HSC depletion, NrasG12D increased HSC proliferation but at the same time increased the self-renewal and competitiveness of HSCs. Serial transplantations revealed that NrasG12D HSCs were able to give higher level of reconstitution than wild-type (WT) HSCs and gave rise to long term multi-lineage reconstitution in lethally irradiated mice after up to four rounds of transplantation while WT HSCs failed to reconstitute beyond two rounds. These effects were not associated with the development of leukemia suggesting oncogenic Nras dys-regulates HSC at a pre-leukemic stage and therefore plays an important role in leukemia initiation. Using histone-2B-GFP (H2B-GFP) label-retaining assays, we further detected a “bimodal” effect of NrasG12D on HSCs: NrasG12D induced a subpopulation of rapid “cycling” HSCs that lost GFP labeling and reconstitution activity faster than WT HSC but another HSC subpopulation that remained more “quiescent” than WT HSCs and retained higher reconstitution when transplanted to irradiated mice. The canonical Ras effector, ERK, was not activated in NrasG12D HSC/Ps and inhibition of ERK with a MEK inhibitor, PD325901, did not have any effect on the Nras induced increase of HSC proliferation. Stat5, on the other hand, was significantly activated in NrasG12D HSC/Ps and heterozygous knockout of Stat5ab abolished the increased proliferation in NrasG12D HSCs, suggesting that Stat5 signaling mediates at least part of the Nras induced increase in HSC proliferation. Nras is thus the first signaling pathway that simultaneously increases HSC proliferation, self-renewal and competitiveness without inducing frank leukemogenesis. This is likely through a “bimodal” effect of Nras signaling on HSC cell cycle regulation. Our studies also identified Stat5 as a novel therapeutic target to inhibit early events in Ras mediated leukemic transformation. Disclosures:No relevant conflicts of interest to declare.

Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras

AbstractThe palmitoylation/depalmitoylation cycle of posttranslational processing is a potential therapeutic target for selectively inhibiting the growth of hematologic cancers with somatic NRAS mutations. To investigate this question at the single-cell level, we constructed murine stem cell virus vectors and assayed the growth of myeloid progenitors. Whereas cells expressing oncogenic N-RasG12D formed cytokine-independent colonies and were hypersensitive to GM-CSF, mutations within the N-Ras hypervariable region induced N-Ras mislocalization and attenuated aberrant progenitor growth. Exposing transduced hematopoietic cells and bone marrow from Nras and Kras mutant mice to the acyl protein thioesterase inhibitor palmostatin B had similar effects on protein localization and colony growth. Importantly, palmostatin B-mediated inhibition was selective for Nras mutant cells, and we mapped this activity to the hypervariable region. These data support the clinical development of depalmitoylation inhibitors as a novel class of rational therapeutics in hematologic malignancies with NRAS mutations.

Abstract 3111: The ETS factor ETV1 is an oncogene in melanoma

Abstract Chromosome 7 copy gains are a recurrent genomic alteration in melanoma. Previous genomic studies identified a common peak of gain within 7p, highlighting it as a region likely to contain “driver” cancer genes. In this study, we identified several melanoma samples with focal amplification events located within this peak, and found that the overlapping region of these amplicons contained the locus of the ETS transcription factor and oncogene, ETV1. Fluorescent in situ hybridization analysis of a progressive melanoma tissue microarray revealed ETV1 amplification in 13% of the primary tumors and 18%of the metastatic melanomas, whereas no amplification in nevus samples. Silencing of ETV1 expression demonstrated dependency on ETV1 expression for proliferation and anchorage-independent growth in some melanoma cell lines. Moreover, over-expression of ETV1 in combination with oncogenic NRASG12D transformed primary melanocytes and promoted tumor formation in mice. Interestingly, overexpression of ETV1 increased MITF expression, a known oncogene in melanoma. Together, these observations suggest an oncogenic role for ETS transcription factors in general and ETV1 in particular in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3111.

An Oncogenic Role for ETV1 in Melanoma

Copy gains involving chromosome 7p represent one of the most common genomic alterations found in melanomas, suggesting the presence of "driver" cancer genes. We identified several tumor samples that harbored focal amplifications situated at the peak of common chromosome 7p gains, in which the minimal common overlapping region spanned the ETV1 oncogene. Fluorescence in situ hybridization analysis revealed copy gains spanning the ETV1 locus in >40% of cases, with ETV1 amplification (>6 copies/cell) present in 13% of primary and 18% of metastatic melanomas. Melanoma cell lines, including those with ETV1 amplification, exhibited dependency on ETV1 expression for proliferation and anchorage-independent growth. Moreover, overexpression of ETV1 in combination with oncogenic NRAS(G12D) transformed primary melanocytes and promoted tumor formation in mice. ETV1 overexpression elevated microphthalmia-associated transcription factor expression in immortalized melanocytes, which was necessary for ETV1-dependent oncogenicity. These observations implicate deregulated ETV1 in melanoma genesis and suggest a pivotal lineage dependency mediated by oncogenic ETS transcription factors in this malignancy.

The RALGEF Pathway Contributes to the Pathogenesis of Chronic Myelogenous Leukemia by BCR/ABL

Selected inhibitors of the BCR/ABL tyrosine kinase have shown a remarkable clinical activity in patients with chronic myelogenous leukemia (CML). However, these drugs do not completely eradicate leukemic cells and drug resistance emerges. Identification of additional contributors to the pathogenesis of CML remains to be important for developing strategies for overcoming resistance to BCR/ABL kinase inhibitors and for eradicating leukemic cells. We and others have previously shown that expression of BCR/ABL in mouse bone marrow cells by retroviral transduction and transplantation (BMT) efficiently induces a CML-like myeloproliferative disorder (MPD). Using this BMT model for CML, we have found that the Grb2 SH2 binding site Y177 of BCR/ABL, which is critical for BCR/ABL to activate the RAS pathway, is required for the induction of CML-like disease by BCR/ABL. We have further shown that oncogenic NRAS (NRASG12D) can rescue the defect of the Y177F mutant BCR/ABL in the induction of CML-like disease. To further elucidate the pathways downstream of NRAS responsible for this “rescue” effect, we utilized three well-characterized NRAS effector mutants, G12D/T35S (activating RAF but is impaired in binding PI3K and RALGEF), G12D/E37G (activating RALGEF but not RAF or PI3K), and G12D/Y40C (activating PI3K but not RAF or RALGEF). None of the three NRAS effector mutants could induce hematological malignancies by themselves, but all were capable of “rescuing” the CML defect of BCR/ABLY177F to some extent. Interestingly, the NRAS G12D/E37G effector mutant was the most efficient. We further examined the role of RALGEF pathway by introducing an activated RALGDSCAAX into our BCR/ABLY177F mouse model. RALGDS-CAAX was able to cooperate with BCR/ABLY177F to induce a CML-like MPD in nearly half of mice whereas RALGDSCAAX alone did not induce diseases in any mice. As a complement to this study, we also introduced dominant negative RAL A or RAL B in the background of BCR/ABL. This resulted in a significant delay in disease onset. Taken together, this data sheds light on the importance of the RALGEF pathway in BCR/ABL-mediated CML induction and supports the notion of inhibition of this pathway as a potential therapeutic strategy.

Biology, Therapy, and Resistance of Myeloid Malignancies Initiated by Hyperactive Ras.

Abstract Ras proteins regulate cell fates by cycling between active GTP-bound and inactive GDP-bound states (Ras·GTP and Ras·GDP). Ras·GTP modulates cell fates by activating effector pathways that include the Raf/MEK/ERK, phosphoinositol 3’-kinase (PI3K)/Akt, and Ral·GDS cascades. Signaling terminates when Ras·GTP is hydrolyzed to Ras·GDP. GTPase activating proteins (GAPs) are negative regulators of Ras output that increase the rate of GTP hydrolysis. Mammalian cells express two major GAPs – p120GAP and neurofibromin. The latter is encoded by the NF1 tumor suppressor gene, which is mutated in persons with neurofibromatosis type 1 (NF1). Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and other cancers. Somatic RAS mutations are also common in myeloid malignancies, and other leukemia-associated mutations, such as FLT3 internal tandem duplications, PTPN11 point mutations, and the BCR-ABL fusion protein deregulate Ras signaling. Together, the prevalence of oncogenic RAS mutations and the existence of these alternative genetic mechanisms establish hyperactive Ras as a major therapeutic target. However, developing inhibitors of oncogenic Ras proteins is extremely challenging due to structural considerations and because an effective drug must restore normal biochemical activity (i.e., repair a broken enzyme). Strains of mice carrying conditional mutant alleles of Nf1, oncogenic Kras, and oncogenic Nras are novel reagents for understanding how cells remodel signaling networks in response to hyperactive Ras and for performing pre-clinical trials. Use of the Mx1-Cre transgene to ablate Nf1 or to activate oncogenic KrasG12D or NrasG12D expression in hematopoietic cells causes myeloproliferative disorders (MPDs) that model JMML and chronic myelomonocytic leukemia (CMML). We are using retroviral insertional mutagenesis (RIM) to identify cooperating mutations that might induce progression from MPD to acute myeloid leukemia (AML). CI-1040, a potent inhibitor of MEK, unexpectedly had no beneficial effects in Nf1 mutant mice with MPD. By contrast, MEK inhibition induced regression of Nf1-deficient AMLs. These AMLs uniformly developed resistance in vivo, despite equivalent biochemical inhibition of the target in paired sensitive and resistant clones. Analysis of retroviral insertions in resistant AMLs revealed outgrowth of a pre-existing clone during CI-1040 administration, and we have implicated RasGRP1 and p38α as modulating resistance in vivo. These data emphasize the importance of cell context in the response to targeted agents and establish a tractable in vivo system for identifying genes that modulate therapeutic efficacy and for probing mechanisms of acquired resistance.