NRASG12V Research Articles

Missense mutation of NRAS is associated with malignant progression in neurocutaneous melanosis

Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated, and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal (VP) shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via VP shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three mutations, namely GNAQ R183Q, S1PR3 G89S and NRAS G12V, considered pathogenic, were found, although S1PR3 mutations have not been previously reported in melanocytic tumors. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites, with copy-neutral loss-of-heterozygosity (CN-LOH) occurring in tumor. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discovering novel driver mutations associated with tumorigenesis and targeted therapy.

10141-GMC-13 MULTIREGIONAL GENOMIC ANALYSIS: A NOVEL APPROACH FOR DETECTING PATHOGENIC DRIVER MUTATION ASSOCIATED WITH MALIGNANT PROGRESSION IN NEUROCUTANEOUS MELANOSIS

Abstract Neurocutaneous melanosis (NCM) is a rare congenital neurocutaneous syndrome characterized by congenital melanocytic nevus of skin and abnormal proliferation of leptomeningeal melanocytes. Early acquisition of post-zygotic somatic mutations has been postulated to underlie the pathogenesis of NCM. The pathogenesis of NCM remains to be fully elucidated and treatment options have not been established. Here, we report for the first time, multiregional genomic analyses in a 3-year-old autopsied girl with leptomeningeal melanomatosis associated with NCM, in which a ventriculo-peritoneal shunt was inserted for the treatment of hydrocephalus. The patient expired six months after the onset due to respiratory failure caused by abdominal dissemination via ventriculo-peritoneal shunt. We performed multiregional exome sequencing to identify genomic differences among brain and abdominal tumors, nevus, and normal tissues. A total of 87 somatic mutations were found in 71 genes, with a significantly large number of gene mutations found in the tumor site. The genetic alterations detected in the nevus were only few and not shared with other sites. Three pathogenic mutations, GNAQ R183Q, S1PR3 G89S and NRAS G12V were found. GNAQ and S1PR3 mutations were shared in both tumor and normal sites. Moreover, the mutant allele frequencies of the two mutations were markedly higher in tumor sites than in normal sites. NRAS mutation was found only in the abdominal tumor and was thought to be responsible for malignant progression in the present case. Multiregional comprehensive genetic analysis may lead to discover novel driver mutations associated with tumorigenesis and targeted therapy.

Calr+ Myelofibrosis/BCR-ABL Chronic Myeloid Leukemia Overlap Syndrome Treated with Asciminib

Introduction Myeloproliferative neoplasms are considered mutually exclusive, and reports of co-occurring mutations are rare. Over the past 10 years, approximately two dozen case reports have been published of co-occurring BCR-ABL positive and CALR positive patients. Here we present the case of a patient who was reported to have myelofibrosis (MF)/myeloproliferative neoplasm (MPN), later developed chronic myeloid leukemia (CML), and ultimately progressed to acute myeloid leukemia (AML). This is unique in the literature reviewed, and may be the first reported case of this overlap syndrome being treated with asciminib. Case presentation A 40-year old male was diagnosed in 2018 with MF/MPN based upon a hypercellular bone marrow biopsy with diffuse reticulin fibrosis. He was negative for JAK2, but was not tested for CALR or MPL mutations. Treatment was ruxolitinib and hydroxyurea. Two years later, the patient was diagnosed with CML after being admitted for recurrent fevers, headache, and shortness of breath. At that time, a type 2 CALR mutation (CALR K385Nfs*47) was noted. He started dasatinib and decitabine, which resulted in a drop from 28.31% BCR-ABL to 0.0196% (Table 1A). However, hyperuricemia and renal failure led to a change in regimen and the patient was started on asciminib and ropeginterferon alfa-2b-njft in January 2022. By December, the patient's BCR-ABL PCR dropped below quantification, but he received a diagnosis of AML based on a bone marrow biopsy which showed 43% blasts. He received cladribine and cytarabine, but was found to have leukemic infiltration of the spinal cord five months later. The patient was referred to hospice and passed away in June 2023. Discussion To our knowledge, this is the first MPN-CML patient reported to have been treated with asciminib. This case illustrates its effectiveness in treating CML, but highlights the need for improved treatments for CALR+ MPN. Moreover, this case illustrates the importance of testing for CALR, MPL, and BCR-ABL in addition to JAK2 immediately when a patient presents with an MPN. It is unknown whether this patient's CALR mutation preceded the BCR-ABL translocation, and there is debate in the literature with respect to mutation chronology. The patient's BCR-ABL burden dropped from 28.31% to 0.0196% while the CALR burden remained around 94% over the same period (Table 1B). Thus, it is unlikely that the BCR-ABL mutation preceded the CALR mutation within the same clone. The patient's >90% CALR allele burden is higher than reported in the other reported cases, where the burden ranged from 10% to 68%. Furthermore, none of the other cases were reported to progress to AML, though follow up data is lacking. The heavy allelic burden in our patient may have been a contributing factor to the development of AML, as has been reported elsewhere. The patient's overall mutational profile is of interest. Type 2 CALR mutations have been reported to have less favorable prognoses than type 1 mutations. In addition to the CALR and BCR-ABL mutations, the patient had ASXL1 A861Hfs*6, EZH2 D730*, and NRAS G12V. All three mutations are associated with unfavorable outcomes in MDS, AML, and/or CML. Genomic instability has been discussed in relation to both CML and JAK2-positive MPN. Our patient's heavy mutational burden suggests CALR-positive neoplasms may also present against a background of inherent genomic instability. Conclusion We reported a case of MPN, followed by CML, and then AML. The patient was positive for CALR and BCR-ABL. He achieved a complete response in CML after targeted treatment, with asciminib consolidating the gains the patient made on earlier therapies. However, he always maintained a high CALR burden. This patient's mutational profile and disease course argue for the importance of early testing for CALR and BCR-ABL in addition to JAK2 and MPL in the setting of an MPN and for further research into the underlying causes of these overlap conditions.

Single Cell Correlation Analysis: A Novel Method to Analyze Single Cell RNA Sequencing Data Identifies a Self-Renewing Subpopulation of Human Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy. Despite early response to standard chemotherapy, 60-70% of patients relapse with treatment resistant disease and die of relapsed disease. Leukemia stem cells (LSCs), the subpopulation of leukemia cells with self-renewal capacity, can recapitulate leukemia to cause relapse. Ongoing work to define therapeutic targets that can block self-renewal would prevent relapse and disease progression in AML. Prior work has established that LSCs express a distinct gene expression profile. We sought to define the gene expression profile of self-renewal at the single-cell level because a precise understanding of the transcriptional basis for self-renewal could provide more effective therapeutic targets. Our group previously demonstrated that activated NRAS (NRAS G12V) mediates self-renewal in the LSC-enriched subpopulation of AML in the Mll-AF9/NRAS G12V mouse model of AML (Sachs 2014). Single cell RNA-sequencing (scRNA-seq) of the LSC-enriched subpopulation identified three distinct transcriptional profiles. Two genes that encode cell surface markers, Cd36 and Cd69, delineate these distinct profiles. Using in vivo leukemia engraftment assays, we found that only the CD36 -CD69 + LSC subgroup repopulates leukemia while the CD36 +CD69 - LSC subgroup does not, demonstrating that self-renewal capacity is limited to the CD36 -CD69 + subset of the LSC-enriched compartment (Sachs 2020). In contrast, the CD36 +CD69 - LSCs were highly proliferative while the CD36 -CD69 + LSCs were poorly proliferative. These experiments show that self-renewal and rapid proliferation are mutually exclusive features of LSCs, as has previously been shown in normal hematopoietic stem cells (Li 2013). Methods and Results We investigated whether primary human AML stem cells express the murine-derived single-cell self-renewal and proliferation gene expression profiles at the single-cell level. Current computational methods to compare single-cell gene expression profiles use arbitrary similarity scores and do not assess the statistical significance of their findings. Therefore, we developed a new computational method called Single cell Correlation Analysis (SCA). SCA is designed to quantitate the expression of a predefined profile in single-cell RNA sequencing data. The quantification of the expression profiles is assessed on a per-cell basis in an unsupervised manner, meaning that SCA does not rely on any pre-sorting or classification of the query cells. The significance of the quantitative metric (i.e. level of expression) is determined by permutation-based false discovery rate testing. SCA can therefore identify cells that express a profile of interest within a single-cell RNA sequencing dataset and incorporates a measure of statistical significance to that assignment. We used SCA to analyze scRNA-seq data from primary human AML samples. We used data from two samples sequenced by our group and previously published data from 16 samples (van Galen 2019). SCA identified cells that express the murine single-cell self-renewal profile in every sample (0.32-11.5%; FDR < 0.25). We sorted two primary human AML samples to isolate LSCs (CD34 +CD38 -) prior to single-cell capture and sequencing. SCA identified cells that express murine LSC self-renewal profile in both of these samples as well (8.9-11.3%; FDR < 0.25). Furthermore, with these analyses, we found that the single-cell profiles of self-renewal and proliferation were mutually exclusive in human LSCs, as they are in murine LSCs and normal HSCs. We performed gene set enrichment analysis (GSEA) and confirmed that the human cells that express the murine single cell self-renewal profile are enriched in previously reported LSC self-renewal gene expression profiles. Conclusion We present SCA, a novel computational method designed to quantitatively assess single-cell transcriptional datasets and evaluate the statistical significance of its results. Using this method, we demonstrate that primary human AML samples express the single-cell profiles of self-renewal that we previously validated experimentally. SCA-based identification of human self-renewing cells will allow us to interrogate novel transcriptional features of these cells in future studies.

Same-Cell Co-Occurrence of RAS Hotspot and BRAF V600E Mutations in Treatment-Naive Colorectal Cancer.

Mitogen-activated protein kinase pathway-activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/KRAS G12C, 95%; KRAS G12D/NRAS G12V, 48%; BRAF V600E/KRAS G12D, 44%; and KRAS G12D/NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability-high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability-high. Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo.We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples.These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Resistance profiles of anaplastic lymphoma kinase tyrosine kinase inhibitors in advanced non–small-cell lung cancer: a multicenter study using targeted next-generation sequencing

IntroductionAnaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib are approved for advanced non–small-cell lung cancer (NSCLC) with ALK rearrangement. However, the mechanisms of resistance remain largely unclear. MethodsThis prospective multicenter study analyzed cell-free DNA (cfDNA) and/or cancer tissues of patients with NSCLC after progression on ALK TKI(s), using targeted next-generation sequencing. Patients’ clinicopathologic characteristics and treatment outcomes were analyzed. ResultsOverall, 88 patients were enrolled; 31 cancer tissues and 90 cfDNA samples were analyzed. Five (16%) ALK mutations (L1196M ×2, I1171T, D1203N, G1269A/F1174L) and 3 possible bypass mutations (NRAS G12V, EGFR R108K, PIK3CA E545K) were found in 32 crizotinib-resistant cancers. Four (22%) ALK mutations (G1128A, G1202R, G1269A, I1171T/E1210K) and 3 possible bypass mutations (KIT D820E, MET E1012∗, EGFR P265_C291del) were found in 18 ceritinib-resistant cancers. Four (17%) ALK mutations (G1202R ×2, W1295C, G1202R/L1196M) and 1 possible bypass mutation (EGFR P753S) were found in 24 alectinib-resistant cancers. Two (11%) ALK mutations (G1202R/G1269A ×2) and 2 possible bypass mutations (BRAF V600E, MET D1246N) were found in 18 lorlatinib-resistant cancers. In patients with simultaneous paired tissue and cfDNA samples (n = 20), mutations were identified in 9 (45%) and 6 (30%) cases, respectively; the concordance rate was 45%. ConclusionsThe mechanisms of ALK TKI resistance were heterogeneous; ALK mutations were found in less than one-third of patients. Compound ALK mutations, which may confer lorlatinib resistance, may occur in crizotinib, ceritinib, and alectinib-resistant lung cancers.

Single-Cell Transcriptional Profiling of Acute Myeloid Leukemia Identifies Prognostic Transcriptional Profiles at Diagnosis

Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. In hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the molecular mechanisms that allow AML to relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a mouse model of AML (Mll-AF9 / NRASG12V, Sachs et al. Blood 2014, Kim et al. Blood 2009). We hypothesize that understanding the NRAS-activated pathways required for self-renewal at the single-cell level can be used to identify clinically relevant features of human AML.We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p < 0.004).In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). We used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these human LSCs preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity).Next, we investigated whether CD36 and CD69 delineate clinically relevant transcriptional subgroups in human AML. We used previously published, clinically annotated gene expression datasets of human AML that include associated survival outcomes data (TCGA, GSE12417 (Metzeler et al. Blood 2008), and GSE6891 (Verhaak et al. Haematologica 2009)). Gene Cluster Expression Summary Score (GCESS, Scott et al. under review) analysis identified gene clusters associated with CD69 and CD36 in our murine single-cell dataset. We found that the CD36 and CD69-associated clusters are conserved in the human transcriptional datasets. More importantly, patients that express either the CD69 or the CD36-associated gene expression profile suffer from shorter survival times in these three, independent human transcriptional datasets.In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo . Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. Finally, we found that the gene expression profiles we identified in our murine single-cell data are associated with poor outcomes in human AML indicating that our single-cell gene expression analyses can be used to identify prognostic patient subgroups. These findings suggest that these gene expression profiles can be used to identify functional transcriptional modules associated with poor outcome in human AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TAMI-28. DIFFERENTIAL MIGRATION MECHANICS AND IMMUNE RESPONSES OF GLIOMA SUBTYPES

Abstract In Glioblastoma (GBM), tumor spreading is driven by tumor cells’ ability to infiltrate healthy brain parenchyma, which prevents complete surgical resection and contributes to tumor recurrence. GBM molecular subtypes, classical, proneural and mesenchymal, were shown to strongly correlate with specific genetic alterations (Mesenchymal: NF1; Classical: EGFRVIII; Proneural: PDGFRA). Here we tested the hypothesis that a key mechanistic difference between GBM molecular subtypes is that proneural cells are slow migrating and mesenchymal cells are fast migrating. Using Sleeping Beauty transposon system, immune-competent murine brain tumors were induced by SV40-LgT antigen in combination with either NRASG12V (NRAS) or PDGFB (PDGF) overexpression. Cross-species transcriptomic analysis revealed NRAS and PDGF-driven tumors correlate with human mesenchymal and proneural GBM, respectively. Similar to human GBM, CD44 expression was higher in NRAS tumors and, consistent with migration simulations of varying CD44 levels, ex vivo brain slice live imaging showed NRAS tumors cells migrate faster than PDGF tumors cells (random motility coefficient = 30µm2/hr vs. 2.5µm2/hr, p &amp;lt; 0.001). Consistent with CD44 function as an adhesion molecule, migration phenotype was independent of the tumor microenvironment. NRAS and human PDX/MES tumor cells were found to migrate faster and have larger cell spread area than PDGF and human PDX/PN tumors cells, respectively, in healthy mouse brain slices. Furthermore, traction force microscopy revealed NRAS tumor cells generate larger traction forces than PDGF tumors cells which further supports our theoretical mechanism driving glioma migration. Despite increased migration, NRAS cohort had better survival than PDGF which was attributed to enhanced antitumoral immune response in NRAS tumors, consistent with increased immune cell infiltration found in human mesenchymal GBM. Overall our work identified a potentially actionable difference in migration mechanics between GBM subtypes and establishes an integrated biophysical modeling and experimental approach to mechanically parameterize and simulate distinct molecular subtypes in preclinical models of cancer.

Leukemogenic Functions of Mutant ASXL1 Are Regulated By CDK-Mediated Phosphorylation

Additional sex combs-like 1 (ASXL1) is recurrently mutated in various myeloid neoplasms and its mutations confer poor prognosis. Most of the ASXL1 mutations in myeloid malignancies are frameshift or nonsense mutations, leading to expression of C-terminally truncated form of ASXL1 (ASXL1-MT). Recently, we have demonstrated that monoubiquitination of ASXL1-MT at lysine 351 plays an important role in enhancing the catalytic activity of BAP1, resulting in reduction of H2AK119ub and upregulation of posterior HOXA genes to promote myeloid leukemogenesis (Asada et al. Nat commun. 2018). However, roles of other post-translational modifications of ASXL1 including phosphorylation remain to be elucidated. In this study, we performed nano liquid chromatography tandem mass spectrometry (nano LC-MS/MS) and found that ASXL1 was phosphorylated at multiple serine (S) and threonine (T) residues including S503 residue, the major phosphorylated site of ASXL1. We generated an antibody that specifically recognizes phosphorylated S503 of ASXL1, and showed that CDK1/2 phosphorylated both wildtype ASXL1 and ASXL1-MT. However, phosphorylation status of ASXL1-MT at serine 503 did not affect its oncogenic activity. Thus, we considered the possibility that phosphorylations of other residues would compensate the effects of phosphorylation of ASXL1-MT at serine 503. Using public database and our data from mass spectrometry, we found additional five S/T residues containing a CDK phosphorylation motif (S/T-P). We then established a non-phosphomimetic mutant form of ASXL1-MT (ASXL1-MT-6A) whose six S/T residues were substituted to alanine. To compare the oncogenic functions between ASXL1-MT-6A and ASXL1-MT, we first assessed the impact of these mutants on differentiation of hematopoietic cells. Expression of ASXL1-MT-6A more strongly impaired differentiation of human CD34+ cord blood (CB) cells towards granulocytes and erythrocytes than that of ASXL1-MT. These effects of ASXL1-MT-6A on differentiation block were also observed in HL-60 cells. Next, we assessed the impact of phosphorylation of ASXL1-MT on its leukemogenic functions using RUNX1-ETO and N-Ras G12V-induced leukemia models, because RUNX1-ETO fusion or N-Ras mutations frequently coexist with ASXL1 mutations in patients with myeloid neoplasms. In agreement with our previous report, expression of ASXL1-MT enhanced proliferation of RUNX1-ETO expressing CB cells. ASXL1-MT-6A showed the stronger growth promoting effect than ASXL1-MT in RUNX1-ETO expressing CB cells in vitro by inhibiting apoptosis and progressing cell cycle. Moreover, ASXL1-MT-6A induced more rapid development of myeloid leukemia driven by N-Ras G12V than ASXL1-MT in vivo. ASXL1-MT-6A induced upregulation of Hoxa5, Hoxa7 and Hoxa9 more strongly than ASXL1-MT in myeloid progenitor cells. These data indicated that ASXL1-MT-6A is an active form of ASXL1-MT. Mechanistically, cycloheximide chasing assay revealed that ASXL1-MT-6A had increased protein stability than ASXL1-MT. Furthermore, cell cycle synchronization and immunoprecipitation assay demonstrated that degradation of ASXL1 protein began at early M phase by interacting with Cdc20, one of the coactivators of anaphase-promoting complex (APC/C), an E3 ubiquitin ligase complex involved in regulating cell cycle progression. Overexpression of Cdc20 promoted degradation of ASXL1, whereas depletion of Cdc20 using CRISPR-Cas9 system increased expression of ASXL1. These biochemical features were also observed for ASXL1-MT. Intriguingly, ASXL1-MT-6A showed resistance to degradation induced by APC/CCdc20. Therefore, CDK-mediated phosphorylation of ASXL1-MT triggers its degradation induced by APC/CCdc20. Collectively, we demonstrated that stability and leukemogenic functions of ASXL1-MT are regulated by CDK-mediated phosphorylation. Dynamics of protein degradation of ASXL1-MT triggered by phosphorylation and ubiquitination could be targets for therapeutic interventions of myeloid neoplasms harboring an ASXL1 mutation. Disclosures No relevant conflicts of interest to declare.

NRASG12V-Mediated Self-Renewal Signaling in Self-Renewing AML Stem Cells.

In acute myeloid leukemia (AML) standard therapies often induce complete remission, but patients frequently relapse and die of the disease. Leukemia stem cells (LSCs) have self-renewal potential and ability to recapitulate the disease. Our goal is to define the molecular mechanisms that allow AML to relapse. We have previously shown that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation in a mouse model of AML (Mll-AF9/NRASG12V, Sachs et al. Blood 2014). We subsequently utilized single-cell RNA sequencing of the LSCs from this model to define and validate the only subset of the LSC-enriched population that can efficiently transplant leukemia in mice. We hypothesize that NRASG12V exerts a unique signaling profile that directs self-renewal in this subset of LSCs. Understanding these pathways at the single-cell level would enable us to design rational therapeutics that would prevent relapse in AML.We used mass cytometry (CyTOF2) to define the signaling activation state of LSC subsets in our AML model. Similar to flow cytometry, mass cytometry provides quantitative measurements of cell-surface and intracellular proteins at the single-cell level. In addition, it can simultaneously and accurately measure over 40 proteins, allowing us to quantitate a panel of intracellular signaling molecules in well-defined immunophenotypic leukemia subpopulations. We previously reported that the LSC-enriched population in this leukemia model is Mac1LowKit+Sca1+ (MKS) and subsequently showed that the self-renewing subset within the MKS population is MKSCD36LowCD69High. In contrast, the MKSCD36HighCD69Low population is incapable of transplanting leukemia in mice.The MKS cells displayed elevated levels of activated signaling molecules relative to the non-MKS population. Comparing the MKS subsets to each other, we found that the self-renewing MKSCD36LowCD69High population displayed significantly higher levels of several signaling molecules including Myc, NF-kB, and β-catenin relative to MKSCD36HighCD69Low cells (which lack self-renewal capacity). We reasoned that self-renewal might be mediated through these signaling molecules uniquely elevated in MKSCD36LowCD69High cells. Next, we sought to define the global signaling activation network within individual MKS subsets to determine if the signaling cascades and dependencies vary between these populations. We used Bayesian network modeling (Sachs K et al. Science 2005) to compare the statistical relationships between these signaling molecules, at the single-cell level. Signaling molecules that impact the levels of downstream effectors can be inferred using this approach. Using this method, we found that the signaling activation network does not significantly vary between MKS subsets. These observations suggest that self-renewal may be driven by alteration in the levels of signaling intermediates rather than alternate signal transduction architecture. We previously found that NRASG12V-mediated signals drive self-renewal in this AML model (Sachs Z. et al. Blood 2014). We used this model to ask which of these self-renewal-associated signaling molecules might be NRASG12V-regulated. We abolished NRASG12V transgene expression in these mice and harvested leukemia cells 72 hours later (per our standard lab protocol). Using this approach, found that self-renewal-associated signaling molecules, including NF-kB and β-catenin, are significantly reduced after NRASG12V-withdrawal indicating that NRASG12V -dependent signaling likely leads to the increase in these signaling molecules.In conclusion, we used mass cytometry analysis to identify the LSC self-renewal-associated signaling state in a murine model of AML and show that NRASG12V activates this signaling program. These data can be used to rationally design therapeutics such as small molecule inhibitors to target self-renewal-specific signaling and prevent relapse in AML. DisclosuresNo relevant conflicts of interest to declare.

Abstract PR09: Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells

Abstract Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the self-renewing LSCs in order to develop therapeutics that target them and eliminate AML relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a transgenic mouse model of AML (Mll-AF9/NRASG12V) (Sachs et al. Blood 2014; Kim et al. Blood 2009). We hypothesize that self-renewal capacity and the NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p &amp;lt; 0.004). In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). In order to determine if the single-cell transcriptional profiles of our murine AML can be found in primary human AML precursors, we performed single-cell RNA sequencing on CD34+ human AML cells obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and they differentially express RAS-activated gene expression profiles and profiles of hematopoietic differentiation. Next, we used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these cells preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity). In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo. Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. These data suggest that single-cell gene expression profiling can delineate leukemia cells with true self-renewal capacity. This abstract is also being presented as Poster 43. Citation Format: Klara E. Noble-Orcutt, Karen Sachs, Connor Navis, Alexandria Hillesheim, Ian Nykaza, Rebecca S. LaRue, Conner Hansen, Ngoc Ha, Michael A. Linden, David A. Largaespada, Zohar Sachs. Single-cell transcriptional profiling of acute myeloid leukemia identifies self-renewing stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR09.

Abstract 3199: Diverse non-FLT3 molecular mechanisms of crenolanib resistance

Abstract FMS-like tyrosine kinase 3 (FLT3) activating mutations are primary molecular targets for the treatment of acute myeloid leukemia (AML) due to high prevalence and unfavorable prognosis. Several type II FLT3 inhibitors have shown clinical benefits but acquisition of secondary FLT3 mutations was reported as a common mechanism of resistance. Previous studies showed that crenolanib, a type I FLT3 inhibitor, had clinical activity without acquisition of secondary FLT3 mutations. To identify the molecular mechanisms associated with crenolanib sensitivity and resistance, we performed exome sequencing on crenolanib treated patients with FLT3-mutant multiply relapsed or refractory AML. Baseline mutational analysis revealed distinct mutational profiles in patients with prior FLT3 inhibitor exposure, especially in the following pathways: NRAS, IDH1, WT1 and RUNX1. Further analysis of patients who had poor response to crenolanib showed mutations in other cell signaling genes such as NRAS and PTPN11. Variant allele frequency (VAF) analysis showed that these mutations sometimes occurred in subclones independent of the FLT3 mutation-bearing clone or sometimes were acquired by the FLT3 mutation-bearing clone. To characterize the influence of these mutations on crenolanib sensitivity, we transduced genes of interest into cell lines harboring FLT3 activating mutations and treated these cells with crenolanib at various concentrations. Significantly increased crenolanib IC50 and IC90 were observed in NRAS G12V MOLM14 and PTPN11 A72D/FLT3 D835 Ba/F3 cells relative to the respective control NRAS WT MOLM14 and PTPN11 WT/FLT3 D835 Ba/F3 cells. Notably, addition of trametinib restored crenolanib sensitivity and demonstrated synergistic cytotoxic effects on cells with FLT3 and NRAS or PTPN11 mutations. In addition, increases in TET2 nonsense/frameshift mutations were observed in patients who did not respond to crenolanib. VAF analysis demonstrated that TET2 mutations co-occurred with FLT3 mutations. We also observed that bone marrow cells from FLT3-ITD knock-in/TET2 knock-out mice are resistant to crenolanib at low concentrations, but remain sensitive to azacytidine at the same level as FLT3-ITD knock-in/TET2 WT cells. The remaining patients exhibited a diverse spectrum of secondary mutations associated with chromatin modifiers, cohesion, spliceosomes and transcription factors which mostly expanded during treatment, suggesting an elaborate genetic/epigenetic mechanism of resistance to crenolanib. Our data suggest that comprehensive sequencing should be carried out on patient samples prior to treatment to identify and pre-emptively target problematic clones. In addition, even with high VAF FLT3 mutations, although FLT3 inhibitor monotherapy provide some clinical benefit, combining agents targeting cooperative lesions will be imperative to eradicate both the dominant clone and resistant subclones and improve patient responses. Citation Format: Haijiao Zhang, Anna M. Schultz, Samantha Savage, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Christopher Eide, Hoang Ho, Yee L. Lam, Richard Sweat, Jaime Faulkner, Evan Lind, Jeffrey W. Tyner. Diverse non-FLT3 molecular mechanisms of crenolanib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2017-3199

PTU-085 A NOTCH1-Driven Secretome Switch Modulates Immune Surveillance In Ras-Induced Senescence

Introduction Oncogene-induced senescence (OIS) is an intrinsic tumour suppressor mechanism, but its impact on tumorigenesis is largely dependent on the nature of the senescence-associated secretory phenotype (SASP). Major components of the SASP include TGFβ1 and pro-inflammatory cytokines, such as IL1, IL6, and IL8, that have pleiotropic context-dependent effects. Methods We utilised the well validated ER:Ras G12V IMR90 HDF in vitro model which undergo Ras-induced senescence (RIS) with 4 OHT. Genetic manipulation was achieved through retroviral gene transfer; transcriptional profiling by mRNA-seq; validation through qPCR and immunoblotting. In vivo hepatocyte senescence was achieved through hydrodynamic tail-vein delivery of NRAS G12V -containing transposons. Results We have demonstrated that NOTCH1, a highly conserved receptor is up-regulated in RIS. In contrast to the up-regulation of NOTCH1, downstream signalling is dynamically regulated: the cleaved, active intracellular domain of NOTCH1 (N1ICD) and NOTCH-target genes were transiently up-regulated at an early phase of RIS, but down-regulated at full senescence. The dynamic expression patterns of N1ICD and TGF-β1 expression were nearly identical, and inversely correlated with the cytokines, IL1 and IL8. Inhibition of NOTCH1 signalling, through expression of a dominant-negative form of the NOTCH1 binding partner MAML1, led to a reduction in TGF-β1, but increased IL1 and IL8 expression during RIS. In addition, ectopic restoration of N1ICD in established RIS cells drove reciprocal secretome changes with reduced IL1 and IL8 and increased TGF-β1, suggesting NOTCH1 signalling plays a critical role in secretome switching. Utilising an NRAS-drive hepatocyte senescence model, NOTCH1 was upregulated during RIS in vivo. Previously it has been shown that the SASP in RIS is critical for the immune-mediated clearance of senescent hepatocytes. Consistent with the enhancement of the pro-inflammatory secretome, co-delivery of the NOTCH inhibitor dnMAML1 with RAS, accelerated the infiltration of CD3+ T-cells into the liver and promoted the clearance of senescent hepatocytes. Restoration of N1ICD in hepatocyte RIS led to senescence bypass and tumour formation. Conclusion The transition to RIS is correlated with a NOTCH1-mediated switch from a TGFβ-rich secretome to a proinflammatory secretome. Promotion of this proinflammatory secretome, through NOTCH inhibition enhances immune surveillance of senescent hepatocytes. This raises the possibility of therapeutically promoting the clearance of senescent cells in vivo. Disclosure of Interest None Declared

Abstract B34: Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature.

Abstract Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Using a murine model of AML that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V), we have found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival (Sachs et al. in revisions). Similarly, recent work has shown that NRASG12V has bimodal, mutually exclusive effects in hematopoietic stem cells (HSCs) providing a proliferation advantage in some HSCs and a self-renewal advantage in others (Li et al. Nature 2013). These data show that mutant NRAS enforces different behaviors in similar cells and suggest that these discrepant effects may be mediated by different NRAS-activated pathways and gene expression pathways. In order to define the mechanisms by which NRASG12V directs self-renewal in AML, we investigated the NRASG12V-mediated transcriptional profile of self-renewing leukemic subgroups and single cells from our AML model. We sorted our primary murine AML into immunophenotypic subgroups and performed bulk cell RNA sequencing on these groups. The Mac-1High group displayed poor self-renewal capacity in colony forming assays and in vivo leukemia transplant assays. In contrast, the Mac-1Low/c-Kit+/Sca-1+ (MLowKS) subgroup was enriched for self-renewal capacity and displayed significantly increased colony forming ability and enhanced in vivo leukemia transplantability. Using principal component analysis and unsupervised hierarchical clustering, we found that each of our immunophenotypic subpopulations were transcriptionally distinct. Using this dataset, we identified the NRASG12V-directed leukemia self-renewal gene expression signature. Gene Set Enrichment Analysis (GSEA, et al. 2005) of this dataset showed that the MLowKS group preferentially expressed previously published transcriptional signatures of leukemia self-renewal. These data also show that NRASG12V enforces distinct gene expression signatures among leukemic subpopulations. Since others have shown that NRASG12V can enforce bimodal behaviors among cells within the same hematopoietic compartment (HSCs, Li et al. Nature 2013), we speculated that the NRASG12V–mediated transcriptional signature critical for leukemia self-renewal may be variable among individual cells. In order to identify that signature and to assess the cell-to-cell heterogeneity of NRASG12V-mediated transcription, we performed single cell RNA sequencing of unsorted and sorted leukemia cells from our murine AML model. In unsupervised hierarchical clustering algorithms, our single cells clustered into five distinct transcriptional signatures, demonstrating that individual leukemia cells are transcriptionally distinct and identifying the NRASG12V-mediated leukemia self renewal signature. Currently, we employ ultra-high parameter flow cytometry (mass cytometry, CyTOF) to identify the cell type specific signaling pathways associated with these discrepant transcriptional signatures. These data identify the NRASG12V-mediated self-renewal gene expression signature at the single cell level and will allow us to identify the specific signaling pathways that activate this signature. Citation Format: Zohar Sachs, Rebecca S. LaRue, Klara Noble, Susan K. Rathe, Aaron L. Sarver, Ngoc A. Ha, David A. Largaespada. Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B34.

Clinical and molecular characterization of refractory BRAF mutant metastatic colorectal carcinoma (mCRC): Vall d’Hebron Institute of Oncology phase I program cohort.

587 Background: BRAF mutation (mt) occurs in 5-8% of mCRC and its role as a negative prognostic factor is well known. The up-front detection of BRAF mt is not standardized beyond the boundaries of research. However, since novel emerging specific therapies are increasingly available in clinical trials, the prior determination of this mutation is now warranted. Methods: From 07/2010 to 01/2014, 581 patients (pts) with advanced refractory mCRCunderwent genetic tumor profiling as part of the VHIO Phase I Trial Molecular Prescreening Program. BRAF mt was determined by Sanger sequencing or multiplex PCR and mass detection (MassARRAY, Sequenom) of 268 frequent hotspots in 25 oncogenes including, KRAS, NRAS, and PIK3CA. PTEN status was also assessed by immunohistochemistry. Results: 581 samples were analyzed detecting BRAF mt in 56 (9.6%). In the BRAF mt population, median age was 61 years (45-80) and 54% had right-sided CRC. Metastatic sites were found at: liver 61%; peritoneum 52%; lung 39%. Median number of standard treatment was 4 (0-8) including cetuximab and/or panitumumab in 73%. BRAF V600E mutation was detected in 53 samples whereas V600K, D594V and D594G were seen in one sample each. PTEN was analyzed in 44.6% of samples resulting in low expression (H-score &lt;50) in 56%. Other co-existing mutations were PIK3KCA H1047R, PIK3CA E545K, NRAS G12V, GNAS R201C and EGFR S768I. 22 pts were enrolled at least in one phase I trial with drugs targeting either BRAF alone (n=12) or combined with anti-EGFR (n=5), MEK (n=2), PI3K (n=1) inhibitors or double MEK/PI3K inhibition (n=2). Partial responses (PR) were seen in 3 pts (2 with BRAF inhibitor and EGFR inhibitor and 1 with BRAF inhibitor as a single agent). Conclusions: BRAF mt mCRC pts in our institution have a similar pattern to other series reported. Notably, PTEN low expression was present in almost half of the samples analyzed and other co-existent mutations in the ERK and PI3K pathways were detected. This observation, coupled with the clinical outcome observed with single agent BRAF inhibition in this population, suggests that these patients may derive greater benefit from dual BRAF and either EGFR, MEK or PI3K inhibition.

Abstract B15: NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia

Abstract Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V). We performed gene expression microarray and RNA sequencing of our AML cells in the presence and absence of NRASG12V. By employing computational approaches to explore NRASG12V-responsive genes in our model, we found that NRASG12V enforced the leukemia self-renewal gene expression signature and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival. We used CyTOF (mass cytometry) for a multiplexed analysis of RAS-dependent signaling intermediates, and found that Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. Using RAS-pathway inhibitors, we found NRASG12V maintained leukemia self-renewal through mTor and Mek pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. Recent work has shown that NRASG12V has bimodal effects in hematopoietic stem cells (Li et al. Nature 2013). To understand the mechanism of these bimodal effects, we have performed single cell RNA sequencing on our AML model. We expect that these analyses will reveal the cell-type specific NRASG12V –mediated mechanisms of leukemia self renewal. Citation Format: Zohar Sachs, Rebecca S. LaRue, Hanh T. Nguyen, Karen Sachs, Nurul Azyan Mohd Hassan, Ernesto Diaz-Flores, Susan K. Rathe, Aaron L. Sarver, Sean C. Bendall, Ngoc A. Ha, Miechaleen D. Diers, Garry P. Nolan, Kevin M. Shannon, David A. Largaespada. NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia. [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 B15. doi: 10.1158/1557-3125.RASONC14-B15

NRASG12V oncogene facilitates self-renewal in a murine model of acute myelogenous leukemia

AbstractMutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we used a murine model that harbors Mll-AF9 and a tetracycline-repressible, activated NRAS (NRASG12V). Using computational approaches to explore our gene-expression data sets, we found that NRASG12V enforced the leukemia self-renewal gene-expression signature and was required to maintain an MLL-AF9– and Myb-dependent leukemia self-renewal gene-expression program. NRASG12V was required for leukemia self-renewal independent of its effects on growth and survival. Analysis of the gene-expression patterns of leukemic subpopulations revealed that the NRASG12V-mediated leukemia self-renewal signature is preferentially expressed in the leukemia stem cell–enriched subpopulation. In a multiplexed analysis of RAS-dependent signaling, Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. NRASG12V maintained leukemia self-renewal through mTOR and MEK pathway activation, implicating these pathways as potential targets for cancer stem cell–specific therapies. Together, these experimental results define a RAS oncogene–driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction.

Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures:Sachs:Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

Abstract 4176: Activated NRAS mediates self-renewal capacity in AML

Abstract NRAS mutations are found in 11-14% of AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), implicating NRAS signaling pathways as potential targets for clinical intervention. However, the specific effects of targeting these pathways on AML physiology are currently unclear. We employ a manipulatable murine model of AML to elucidate these phenomena for therapeutic applications. This model harbors a tetracycline repressible, activated NRAS (NRASG12V) transgene along with an Mll/AF9 transgene. Primary leukemia cells are then transplanted into SCID mice and, upon development of clinically evident leukemia, NRASG12V transgene expression is repressed with doxycycline. NRASG12V repression leads to apoptosis and disease remission (Kim et al. Blood 2009). To dissect the critical signaling networks directing remission, immunophenotypic markers, along with the phosphorylation status of critical signaling intermediates, were analyzed by flow cytometry after NRASG12V repression. This analysis revealed that NRAS withdrawal led to an enrichment of less differentiated Mac1/Gr1 negative cells indicating that, in addition to well-described effects on proliferation, NRAS affects leukemia differentiation status. Furthermore, we only found modifications in known NRAS signaling effectors in the less differentiated, Mac1 negative leukemia cells. Therefore, in addition to affecting the differentiation status of the leukemia, oncogene withdrawal leads to cell-type specific signaling alterations. We hypothesize that these signaling alterations mediate remission-induction by altering the self-renewal capacity of the leukemia at the gene expression level. Parallel RNA sequencing and gene expression microarrays were performed to address this hypothesis and revealed that the gene expression pattern of NRASG12V-expressing cells is enriched with hematopoietic self-renewal genes (Krivtsov et al. Nature 2006, Ivanova et al. Science 2002, Gene Set Enrichment Analysis per Subramanian et al. PNAS 2005). Withdrawal of the oncogene led to loss of this self-renewal gene expression program and an enrichment of genes expressed in terminally differentiated, mature cells. Furthermore, the NRASG12V-dependent gene expression program mimics the Mll/AF9-determined, Myb-mediated self-renewal program seen in a similar AML model (Zuber et al. Genes &amp; Dev 2011). These data suggest that the Mll/AF9-mediated self-renewal program requires the continued expression of NRASG12Vand support a novel role for NRASG12V in maintaining the self-renewal capacity AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4176. doi:1538-7445.AM2012-4176