Activating RAS Mutations Research Articles

Trametinib Sensitivity is Defined by a Myeloid Differentiation Profile in Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML) is a common blood cancer marked by heterogeneity in disease and diverse genetic abnormalities. Additional therapies are needed as the 5-year survival remains below 30%. Trametinib is a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor that is widely used in solid tumors and also in tumors with activating RAS mutations. A subset of patients with AML carry activating RAS mutations; however, a small-scale clinical trial with trametinib showed little efficacy. Here, we sought to identify transcriptomic determinants of trametinib sensitivity in AML. We tested the activity of trametinib against a panel of tumor cells from patients with AML ex vivo and compared this with RNA sequencing (RNA-Seq) data from untreated blasts from the same patient samples. We then used a correlation analysis between gene expression and trametinib sensitivity to identify potential biomarkers predictive of drug response. We found that a subset of AML tumor cells were sensitive to trametinib ex vivo, only a fraction of which (3/10) carried RAS mutations. On the basis of our RNA-Seq analysis we found that markers of trametinib sensitivity are associated with a myeloid differentiation profile that includes high expression of CD14 and CLEC7A (Dectin-1), similar to the gene expression profile of monocytes. Further characterization confirmed that trametinib-sensitive samples display features of monocytic differentiation with high CD14 surface expression and were enriched for the M4 subtypes of the FAB classification. Our study identifies additional molecular markers that can be used with molecular features including RAS status to identify patients with AML that may benefit from trametinib treatment.

Involvement of tumor immune microenvironment metabolic reprogramming in colorectal cancer progression, immune escape, and response to immunotherapy.

Metabolic reprogramming is a k`ey hallmark of tumors, developed in response to hypoxia and nutrient deficiency during tumor progression. In both cancer and immune cells, there is a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, also known as the Warburg effect, which then leads to lactate acidification, increased lipid synthesis, and glutaminolysis. This reprogramming facilitates tumor immune evasion and, within the tumor microenvironment (TME), cancer and immune cells collaborate to create a suppressive tumor immune microenvironment (TIME). The growing interest in the metabolic reprogramming of the TME, particularly its significance in colorectal cancer (CRC)-one of the most prevalent cancers-has prompted us to explore this topic. CRC exhibits abnormal glycolysis, glutaminolysis, and increased lipid synthesis. Acidosis in CRC cells hampers the activity of anti-tumor immune cells and inhibits the phagocytosis of tumor-associated macrophages (TAMs), while nutrient deficiency promotes the development of regulatory T cells (Tregs) and M2-like macrophages. In CRC cells, activation of G-protein coupled receptor 81 (GPR81) signaling leads to overexpression of programmed death-ligand 1 (PD-L1) and reduces the antigen presentation capability of dendritic cells. Moreover, the genetic and epigenetic cell phenotype, along with the microbiota, significantly influence CRC metabolic reprogramming. Activating RAS mutations and overexpression of epidermal growth factor receptor (EGFR) occur in approximately 50% and 80% of patients, respectively, stimulating glycolysis and increasing levels of hypoxia-inducible factor 1 alpha (HIF-1α) and MYC proteins. Certain bacteria produce short-chain fatty acids (SCFAs), which activate CD8+ cells and genes involved in antigen processing and presentation, while other mechanisms support pro-tumor activities. The use of immune checkpoint inhibitors (ICIs) in selected CRC patients has shown promise, and the combination of these with drugs that inhibit aerobic glycolysis is currently being intensively researched to enhance the efficacy of immunotherapy.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both invitro and invivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Abstract PR01: Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues

Abstract RAS mutations occur in approximately 20% of human cancers including the majority of pancreatic ductal adenocarcinoma (PDAC), half of colorectal cancers, and a third of all lung cancers. In cases of oncogenic activating RAS mutations, GTP hydrolysis is impaired, and the protein is preferentially held in the active GTP-bound state. When activated, RAS triggers multiple proliferative signaling cascades, including the MAPK/ERK and PI3K pathways, to induce cell growth, division, and differentiation. In solution, RAS exists in two interconverting GTP-bound conformational states, State 1 (inactive) and State 2 (active). State 2 is the active conformation since it binds to RAS Binding Domain (RBD) of effector proteins. Though the State 1 conformation is GTP-bound, it is unable to bind effectors and represents an inactive KRAS conformation. This State 1/State 2 equilibrium is closely coupled to the conformations (γ1 and γ2) exhibited by the γ-phosphate of GTP and its analogues. Solution-state 31P NMR spectroscopy captures and quantifies the γ1 and γ2 peaks representing State 1 and State 2 (Spoerner M et al., JBC 2010). We have initiated solution-state NMR investigation to determine the structural differences between wild-type (WT) and oncogenic mutant KRAS proteins (G12C, G12D, G12V, Q61L, and G13D) when bound to GTP and GppNHp. We quantified the State 1/State 2 equilibrium using 31P NMR. Our results show that GppNHp-bound KRAS proteins harboring oncogenic mutations differentially modulate the State 1/State 2 conformational equilibria resulting in remarkable divergence in conformational populations compared to WT protein; a notion for presence of mutation induced structural plasticity. Much work on the structural biology, biophysics and biochemistry of KRAS proteins is done when bound to the non-hydrolysable GTP analogue, GppNHp. Strikingly, our 31P NMR results show that GTP-bound KRAS elicits significant variations in State 1 and State 2 populations in comparison to GppNHp-bound proteins. These data suggest GppNHp binding induces a significant increase in the State 1 population; an observation that does not translate into GTP-bound KRAS. Our results provide additional insights into the differential binding affinities seen between WT/mutant KRAS and effector proteins (e.g., Hunter JC et al., Mol Cancer Res 2015) as well as interpretation for State 1 conformations in KRAS crystal structures when bound to mostly GppNHp and not GTP. We have also applied this 31P NMR approach to the evaluation of KRAS small molecule inhibitors that shift the population equilibrium to the State 1 (inactive) conformation in the protein-ligand complex. This demonstrates an intriguing therapeutic opportunity and novel mechanism of action that we are further exploring as part of our drug discovery pipeline. Citation Format: Alok K. Sharma, Marcin Dyba, Dominic Esposito, Bin Wang, Pedro J. Beltran, Eli Wallace, Dwight Nissley, Frank McCormick, Anna E. Maciag. Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR01.

Molecular Profiling of Syringocystadenocarcinoma Papilliferum Reveals RAS-Activating Mutations.

Syringocystadenocarcinoma papilliferum (SCACP) is a rare adnexal carcinoma and the malignant counterpart of syringocystadenoma papilliferum (SCAP), which is commonly located on the head and neck and may arise in association with a nevus sebaceus. RAS mutations have been identified in both SCAP and nevus sebaceus. To evaluate the clinicopathologic and molecular features of SCACPs, which have not been previously explored. We obtained 11 SCACPs from 6 institutions and reviewed the clinicopathologic features. We also performed molecular profiling using next-generation sequencing. The cohort comprised 6 women and 5 men with ages ranging from 29 to 96 years (mean, 73.6 years). The neoplasms occurred on the head and neck (n = 8; 73%) and extremities (n = 3; 27%). Three tumors possibly arose in a nevus sebaceus. A total of 4 cases showed at least carcinoma in situ (adenocarcinoma, n = 3; squamous cell carcinoma [SCC], n = 1), and 7 cases were invasive (SCC, n = 5; mixed adenocarcinoma + SCC, n = 2). A total of 8 of 11 cases (73%) had hot spot mutations consisting of HRAS (n = 4), KRAS (n = 1), BRAF (n = 1), TP53 (n = 4), ATM (n = 2), FLT3 (n = 1), CDKN2A (n = 1), and PTEN (n = 1). All 4 cases with HRAS mutations occurred on the head and neck, whereas the KRAS mutation occurred on the extremity. RAS-activating mutations were detected in 50% of the cases, of which most (80%) involved HRAS and occurred on the head and neck, which shows overlapping features with SCAP, supporting that a subset may arise as a result of malignant transformation and likely an early oncogenic event.

Abstract 2425: Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation

Abstract The majority of breast cancer patient deaths occur when tumor cells migrate away from the primary tumors and disseminate metastatically. Cancer cells at the invasive front mimic the behaviors of non-tumorigenic epithelial cells at wound edges but show less coordinated migration comparatively. While most wound healing studies use a timeframe of 24-48 hours, our previous studies have shown that ATP released from wounded cells initiates a calcium (Ca2+) signal within seconds that spreads to neighboring cells through activation of the P2Y2 surface receptor. RNAseq data of MCF10A mammary epithelial cells with stepwise mutations of PTEN knockout and KRas activation alone or in combination, demonstrate that P2Y2 is downregulated specifically by active KRas. Fluorescence microscopy was used to measure Ca2+ signaling and mitochondrial membrane potential in parental and mutant MCF10A cells. Change in total fluorescence was measured and compared to baseline using Fluo-4, a Ca2+ fluorescent indicator, and TMRM (tetramethylrhodamine, methyl ester), a fluorescent mitochondrial membrane potential dye on Nikon analysis software. We show that KRas activation disrupts ATP stimulation and Ca2+ signaling. This change was further quantified using a FlexStation III plate reader with Fluo-4 to read specific fluorescent changes in Ca2+ after ATP addition. ATP-stimulated Ca2+ was also disrupted in other breast tumor cell lines harboring Ras activating mutations, MDA-MB-231 and MDA-MB-436, both showing a similar decrease in P2Y2 expression. These data show that ATP stimulation can be used to further understand the differences in Ca2+ signaling and mechanical-response between normal breast epithelial cells and cancer cells. Clarifying these molecular mechanisms could reveal targets for new cancer treatments, especially since 90% of human tumors are epithelial carcinomas. Citation Format: Makenzy Mull, Stephen Pratt, Rachel Lee, Abanoub Gad, Katarina Chang, David Annis, Keyata Thompson, Michele Vitolo, Liron Boyman, Stuart Martin. Mechano-response via ATP alters calcium signaling in breast epithelial cells with oncogenic KRas mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2425.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells

BackgroundDevelopment of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations.MethodsGenome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled.ResultsBoth genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition.ConclusionsOur results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Epigenetic downregulation of Socs2 contributes to mutant N-Ras-mediated hematopoietic dysregulation.

ABSTRACTRAS mutations occur in a broad spectrum of human hematopoietic malignancies. Activating Ras mutations in blood cells leads to hematopoietic malignancies in mice. In murine hematopoietic stem cells (HSCs), mutant N-RasG12D activates Stat5 to dysregulate stem cell function. However, the underlying mechanism remains elusive. In this study, we demonstrate that Stat5 activation induced by a hyperactive Nras mutant, G12D, is dependent on Jak2 activity. Jak2 is activated in Nras mutant HSCs and progenitors (HSPCs), and inhibiting Jak2 with ruxolitinib significantly decreases Stat5 activation and HSPC hyper-proliferation in vivo in NrasG12D mice. Activation of Jak2-Stat5 is associated with downregulation of Socs2, an inhibitory effector of Jak2/Stat5. Restoration of Socs2 blocks NrasG12D HSC reconstitution in bone marrow transplant recipients. SOCS2 downregulation is also observed in human acute myeloid leukemia (AML) cells that carry RAS mutations. RAS mutant AML cells exhibited suppression of the enhancer active marker H3K27ac at the SOCS2 locus. Finally, restoration of SOCS2 in RAS mutant AML cells mitigated leukemic growth. Thus, we discovered a novel signaling feedback loop whereby hyperactive Ras signaling activates Jak2/Stat5 via suppression of Socs2.

Targeting farnesylation as a novel therapeutic approach in HRAS-mutant rhabdomyosarcoma

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

Abstract GS3-03: Genomic analysis of 733 HER2+ breast cancers identifies recurrent pathways alterations associated with anti-HER2 resistance and new therapeutic vulnerabilities

Abstract Background:The introduction of anti-HER2 therapies, trastuzumab and pertuzumab (HP) to the treatment of metastatic HER2-positive breast cancer has had a transformational impact on the outcomes for this disease. Nevertheless, disease progression is ultimately observed in most patients within 3 years carrying the potential for morbidity and potentially more toxic therapies. To identify selective strategies to prevent disease progression, we sought to elucidate mechanisms of resistance to anti-HER2 therapies by analyzing a large cohort of genomically and clinically annotated HER2+ breast cancers. Methods: Patients with advanced HER2+ breast cancer who underwent prospective clinical tumor sequencing utilizing MSK-IMPACT assay between April 2014 and February 2021 were included in the analysis. Clinical HER2 positivity was defined as per ASCO/CAP guidelines. Cox proportional hazard models were used to determine the association between genomic alterations and progression-free survival (PFS) on 1st line HP and taxane-based therapy (THP). Only patients with a sequenced pre-treatment tumor sample were included in the survival analysis. Recurrent mutations identified were modeled in HER2+ breast cancer cell lines using short hairpin RNAs and CRISPR/Cas9, and the sensitivity of these isogenic pairs to HER2-targeted therapies was evaluated via metabolic and colony formation assays of cell proliferation. Results: We identified 733 ERBB2-amplified primary (n=385) and metastatic (n=348) that underwent sequencing. Concurrent PIK3CA mutations were identified in 30% of the tumors. Pathogenic activating alterations involving the MAPK pathway were observed in 12.8% of tumors with the most frequent alterations being NF1 loss, ERBB2 and RAS activating mutations. MAPK alterations were significantly enriched in the metastatic tumors (16.6%) compared to the treatment-naïve primaries (9.8%, p=0.020). The outcome analysis included 145 patients with advanced clinically HER2+ breast cancer whose tumors were sequenced prior to starting 1st line THP. Twenty percent (29/145) of tumors did not show genomic ERBB2 amplification as detected by NGS and had a significantly worse outcome (median PFS of 9.4 months [95% CI 5.5-19] and 23 months [95% CI 17-30], respectively; p=0.015). PIK3CA mutations were also associated with a shorter PFS (mutant: 13 months [95%CI: 7.7-18] vs wild type: 23 months [95%CI 17-16], p=0.0013). We further found reduced PFS in MAPK altered tumors (median PFS 9.9 months; 95% CI: 5.5-17) compared to the rest of the population (median PFS 21 months; 95% CI: 17-30; p=0.01). On multivariable analysis adjusted for estrogen receptor status, and presence of PIK3CA/AKT1/PTEN mutations and genomic ERBB2 amplification, MAPK pathway alterations were independently associated with worse outcome (HR: 2.25; 95% CI: 1.29, 3.93; multivariate p = 0.0043). To establish a causal role for MAPK alterations in reducing efficacy of anti-HER2 therapy, we depleted NF1 expression or expressed mutant KRAS or BRAF in a panel of HER2+ breast cancer cell lines. Consistently, MAPK-altered cell lines exhibited resistance to FDA approved HER2 inhibitors in vitro and in vivo. Conclusions: This clinicogenomic analysis of mechanisms of resistance to anti-HER2 therapy demonstrated that PIK3CA activating mutations and lack of genomic ERBB2 amplification as detected by tumor sequencing are associated with shortened PFS on HP-based therapy. Our analysis uniquely identified MAPK pathway alterations as additional potential drivers of resistance to anti-HER2 therapy. Inhibition of the PI3K or MAPK pathway in such tumors may represent a new therapeutic strategy to extend H/P benefit. Citation Format: Emanuela Ferraro, Alison Smith, Anton Safonov, Paulino Tallon De Lara, Cristina Bernado', Enrique J. Arenas Lahuerta, Joaquín Arribas, David Solit, Jorge S. Reis-Filho, Neal Rosen, Larry Norton, Shanu Modi, Mark E. Robson, Chau T. Dang, Giuseppe Curigliano, Sarat Chandarlapaty, Pedram Razavi. Genomic analysis of 733 HER2+ breast cancers identifies recurrent pathways alterations associated with anti-HER2 resistance and new therapeutic vulnerabilities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-03.

MZF1 mediates oncogene-induced senescence by promoting the transcription of p16INK4A.

Oncogene induced senescence is a tumor suppressing defense mechanism, in which the cell cycle-dependent protein kinase (CDK) inhibitor p16INK4A (encoded by the CDKN2A gene) plays a key role. We previously reported that a transcriptional co-activator chromodomain helicase DNA binding protein 7 (CHD7) mediates oncogenic ras-induced senescence by inducing transcription of the p16INK4A gene. In the current study, we identified myeloid zinc finger 1 (MZF1) as the transcriptional factor that recruits CHD7 to the p16INK4A promoter, where it mediates oncogenic ras-induced p16INK4A transcription and senescence through CHD7, in primary human cells from multiple origins. Moreover, the expression of MZF1 is induced by oncogenic ras in senescent cells through the c-Jun and Ets1 transcriptional factors upon their activation by the Ras-Raf-1-MEK-ERK signaling pathway. In non-small cell lung cancer (NSCLC) and pancreatic adenocarcinoma (PAAD) where activating ras mutations occur frequently, reduced MZF1 expression is observed in tumors, as compared to corresponding normal tissues, and correlates with poor patient survival. Analysis of single cell RNA-sequencing data from PAAD patients revealed that among the tumor cells with normal RB expression levels, those with reduced levels of MZF1 are more likely to express lower p16INK4A levels. These findings have identified novel signaling components in the pathway that mediates induction of the p16INK4A tumor suppressor and the senescence response, and suggested that MZF1 is a potential tumor suppressor in at least some cancer types, the loss of which contributes to inactivation of the p16INK4A/RB pathway and disruption of senescence in tumor cells with intact RB.

Small Molecule Inhibition of PDE6D-RAS Interaction Suppresses the Growth of Acute Lymphoblastic Leukemia Cell Lines

Small Molecule Inhibition of PDE6D-RAS Interaction Suppresses the Growth of Acute Lymphoblastic Leukemia Cell Lines

Microrna-211 - a Novel Oncogene in Acute Myeloid Leukemia

MicroRNAs are a class of small, non-coding RNA molecules that regulate gene expression by post-transcriptional silencing and regulate many critical cellular processes. In cancer, microRNAs are emerging as important biomarkers of disease and a subset of microRNAs have well-established roles as drivers of cancer development. MicroRNA-211 (miR-211) is an intronic microRNA encoded on human chromosome 15, and deregulated miR-211 expression has been previously demonstrated in melanoma and a range of other cancers. A role for miR-211 in the hematopoietic system, and/or in the development of acute myeloid leukemia (AML) however remains unexplored.Using microRNA expression profiling, across two independent pediatric AML cohorts, we are the first to identify that miR-211 expression is elevated in a subset of patients, from more than 2-fold and up to 50-fold above non-leukemic remission controls. High expression of miR-211 tends to be clustered in patients with cytogenetically normal and M5 AML subtypes. Using hematopoietic reconstitution experiments in mouse models, we show for the first time that enforced expression of miR-211 alone in hematopoietic stem cells is sufficient to drive a partially-penetrant myeloproliferative disease that phenotypically resembles acute myeloid leukemia. Affected mice have an expansion of immature myeloid cells, expressing CD11b and Gr-1 cell surface markers, with monoblastic cellular morphology. This is accompanied by severe anemia and thrombocytopenia, and dissemination of immature hematopoietic cells into the spleen, liver and extramedullary spaces of the bone marrow. We have also explored the potential co-operation between miR-211 and AML oncogenes. Strikingly, preliminary data suggests miR-211 can co-operate with activating RAS mutations to immortalize cells in vitro, and drive AML in vivo .To further explore the molecular mechanisms of miR-211 in AML, we used RNA sequencing and differential expression analysis to compare transcriptional changes induced by miR-211 overexpression in disease mice relative to empty vector controls. We have identified Lin28a as the most downregulated miR-211 gene target. Lin28a is a well-established target of miR-125b in AML and its knockdown leads to an analogous expansion of myeloid cells in vivo as observed in miR-211-dependent AML.Collectively, our results demonstrate a previously unidentified role for miR-211 as an oncogenic microRNA in the development of AML and suggests that miR-211 may potentially drive the development of AML by blocking myeloid differentiation through the repression of Lin28a . DisclosuresNo relevant conflicts of interest to declare.

Molecular and Functional Characterization of Disease-Propagating Stem Cells in Juvenile Myelomonocytic Leukemia

Juvenile Myelomonocytic Leukemia (JMML) is a rare, poor prognosis childhood leukemia characterized by aberrant proliferation of dysplastic myeloid cells and germline or somatic RAS-activating mutations. The only curative treatment is hematopoietic stem cell (HSC) transplantation, however, post-transplant relapse is frequent (~35%) and long-term disease-free survival is ~64%. Approximately half of patients show evidence of clonal evolution with acquisition of additional abnormalities, e.g. mutations of epigenetic/spliceosome components which confer a particularly adverse prognosis. The cellular hierarchy in JMML is poorly characterized with little known about the identity of leukemia stem cells (LSCs), a crucial step towards understanding disease evolution and propagation of relapse.We therefore set out to functionally and molecularly characterize LSCs in a cohort of 16 JMML patients enrolled in the UK NIHR Paediatric MDS/JMML study with samples at diagnosis, post-transplant and relapse. All patients underwent genetic analysis using a novel targeted next generation sequencing (NGS) panel confirming typical RAS pathway mutations in all patients and additional mutations in epigenetic/spliceosome genes in 44% of the patients. Based on adult MDS/MPN, we hypothesized that the CD34+ HSC and progenitor cell compartment (HSPC) would be preserved in JMML and that JMML-LSCs may reside within the phenotypic HSC compartment (Lin-CD34+CD38-CD90+CD45RA-). However, FACS analysis revealed disruption of HSPCs with a 2 fold reduction in phenotypic HSCs (Lin-CD34+CD38-CD90+CD45RA-) and an aberrant Lin-CD34+CD38-CD90+CD45RA+ (90+/45RA+) population not present in normal pediatric bone marrow (BM). In single cell and bulk in vitro assays, JMML-HSPCs had myeloid-biased lineage output with reduced and dysplastic erythroid and severely impaired megakaryocyte and lymphoid potential. Using a novel, integrated single cell genotyping technique combining HSPC index-sorting with clonogenic assays we backtracked patient-specific mutations to HSPC populations, establishing that: 1. all somatic mutations could be backtracked to the phenotypic HSC compartment (492 colonies genotyped for 9 different mutations in 4 patients); and 2. clonal evolution events (eg ASXL1 mutation) in JMML occurred within the HSC compartment with RAS pathway mutations as a “first hit”. Moreover, a PTPN11 mutation could be traced back to cells within the HSPC compartment of a post-BMT patient prior to molecular evidence of overt disease relapse in whole BM.We next assessed the stem cell capacity of FACS-purified phenotypic HSPCs in JMML by in vitro long-term culture initiating cell (LTC-IC) assays and in vivo xenotransplantation in NSG mice. LTC-IC assays consistently demonstrated self-renewal capacity in HSCs together with variable LTC potential in the novel 90+45RA+ population. Similarly, transplantation of purified JMML HSCs resulted in higher engraftment at 5, 10, and 16 weeks than 90+45RA+ or GMP populations (16 weeks post BMT: HSC = 91.7% human CD45; 90+/45RA+ = 0.14%; GMP = 0.2%, p < 0.0005 for HSC vs GMP, n = 3). Importantly, mice transplanted with HSCs, but not other HSPC populations, developed myeloid leukemia with 67% mortality (p < 0.01, n = 6). Secondary transplantation of HSCs confirmed LT potential of JMML HSCs.Single cell RNA-seq on Lin-CD34+ HSPCs using the Chromium 10X platform (n > 10,000 single cells) showed co-clustering of JMML and normal cells transcriptionally resembling myeloid progenitors expressing MPO and MS4A3, while a distinct cluster of JMML HSPCs uniquely co-expressed fetal genes (e.g. HMGA2), stem cell transcription factors (e.g. GATA2) and myeloid leukemia-associated genes (e.g HEMGN, HOPX, MEF2C). RNA-seq of FACS-purified JMML HSCs also showed upregulation of stem cell and fetal genes (HLF, MEIS1, ABCB1, MPL, and HMGA2) as well as candidate therapeutic targets e.g. MTOR, PIM1, SLC2A1, and CD96 compared to normal HSCs. We confirmed aberrant expression of CD96 in JMML HSCs by flow cytometry, and experiments inhibiting therapeutic targets in vitro and in vivo are underway. In conclusion, we show that JMML HSPCs are organized as a hierarchy, with counterparts of phenotypic HSCs representing disease propagating LSCs. Single cell molecular profiling of JMML HSCs has identified a number of novel potential therapeutic candidates for future clinical development. Disclosuresde la Fuente:bluebird bio: Consultancy; HCA: Consultancy; Jazz Pharmaceuticals: Honoraria. Mead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

Genomic profile of columnar cell variant of papillary thyroid carcinoma

Columnar cell variant (CCV) is a rare papillary thyroid carcinoma subtype. The majority of CCV occur in older patients and are large, invasive tumours that pursue an aggressive clinical course. Rare well-circumscribed CCV occur in younger female patients and are comparatively indolent. We retrospectively identified CCV with material available to perform targeted next-generation sequencing and correlated molecular results with clinicopathological features and outcome. Our cohort was comprised of nine CCV. Nearly all were aggressive tumours; however, one was predominantly well-circumscribed and arose in a thyroglossal duct cyst of a 26-year-old woman who had no evidence of disease at last follow-up. Seven (78%) cases demonstrated activating oncogenic driver alterations in BRAF, including BRAF V600E, an activating N486_P490del deletion, and BRAF-AGK fusions. Activating RAS mutations were seen in two (22%) cases. Additionally, three (33%) cases had TERT promoter mutations, four (44%) had loss of the tumour suppressor CDKN2A and one (11%) case had a loss of function TP53 mutation. Most cases (89%) also demonstrated copy number alterations, including recurrent gain of chromosome 1q (five cases) and losses of chromosome 9p (three cases) and 22q (four cases). The one case without secondary pathogenic mutations or copy number alterations was the tumour in the 26-year-old woman. We found that CCV is primarily a BRAF-driven tumour, with most also harbouring secondary oncogenic mutations and multiple chromosomal gains and losses. Moreover, our findings suggest that molecular analysis could potentially be used to help risk stratify CCV.

Selected Articles from This Issue

Angiosarcomas (AS) are rare, aggressive tumors arising from vascular tissues, and harbor extensive genomic events such as driver gene mutations and chromosomal translocations. However, recurrent fusion products and specific genomic events that promote disease progression have thus far proven elusive. In this study, Kim and colleagues employ RNA sequencing data from human angiosarcomas and canine hemangiosarcomas—which closely mirror human AS—and identify 25 novel fusion products among the human and canine samples. Though the fusions were themselves non-recurrent, they co-occured alongside recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS, suggesting that genomic instability induced by these mutations may play a role in the genomic complexity of the disease. Further, the novel fusions were uniformly associated with increased angiogenic signaling, indicating that diverse genomic events in AS likely converge on the activation of a specific transcriptional program. Taken together, the data nominate a host of new molecular targets for investigation and clarify a unifying role for diverse genomic events in the pathogenesis of AS.Extracellular vesicles (EV) have emerged as a key mediator of intercellular signaling and have been linked to the pathogenesis of cancer. EV are loaded with different “cargo” depending on their source, and the characterization of these cargoes is important to understanding and contextualizing their role. Here, Ikeda and colleagues use proteomics to quantitatively probe the differences between EV from cancerous versus normal colon. Employing an ex vivo method for harvesting tissue-exudative EV (Te-EV) from patient samples, the authors find that the amino acid transporter CAT1 is enriched in Te-EV from colorectal cancer (CRC). CAT1-positive EV were found at higher abundance in the plasma of CRC patients and were shown to mediate CRC pathogenesis: by modulating the metabolism of arginine and nitric oxide in endothelial cells, CAT1-positive EV from CRC tissues promoted vascular cell proliferation and tubule formation. These observations identify a key mechanism underlying angiogenesis in CRC and nominate CAT1-positive EV as a potential plasma biomarker for CRC development and progression.Oncogene signaling is a potent driver of tumorigenesis and progression, but cells inherently resist oncogenic transformation via feedback loops leading to cellular senescence. Understanding how oncogene-induced senescence (OIS) is subverted by tumors is critical to the development of new methods to clinically manage cancers driven by BRAF- and RAS-activating mutations. Here, Garnett and colleagues employ a genetic screen to identify mediators of OIS in the context of BRAF activation. The authors found that Amylin, a 37-amino acid peptide that promotes glycolysis, is a key molecular determinant of the response to oncogene signaling. Amylin-ablated cells underwent a series of metabolomic and epigenetic changes that circumvented the onset of senescence in response to BRAF signaling. However, exogenous supplementation of Amylin to the cell growth medium restored the ability of cells to enter a senescent state. These data identify Amylin as a key control node governing the response to oncogene signaling in cancer cells, and suggest that Amylin treatment may hold clinical benefit for patients with oncogene-driven cancers.Acute myeloid leukemia (AML) is a genetically diverse cancer with over ten different molecular subtypes characterized by specific genomic events. EGFR pathway signaling has also been described in AML cells, but most AML cells paradoxically do not express any EGFR family members on the cell surface. In this study, Kam and colleagues address this discrepancy and identify intracellular expression of truncated ERBB2 isoforms in AML cells and myelodysplastic syndrome, an AML precursor lesion. These ERBB2 isoforms—which are also known to be pathogenic in breast cancer—mediated active signaling via classical EGFR-related pathways and were sensitive to inhibition with ERBB2 tyrosine kinase inhibitors. While ERBB2 inhibition was effective as a single agent, combination of the ERBB2 inhibitor lapatinib with the BCL-2 inhibitor venetoclax resulted in marked synergism at all tested doses. These observations nominate ERBB2 inhibitors for further investigation to complement the growing arsenal of therapeutic options for AML patients.

GNA14, GNA11, and GNAQ Mutations Are Frequent in Benign but Not Malignant Cutaneous Vascular Tumors.

Cutaneous vascular tumors consist of a heterogeneous group of benign proliferations, including a range of hemangiomas and vascular malformations, as well as heterogeneous groups of both borderline and malignant neoplasms such as Kaposi’s sarcoma and angiosarcomas. The genetics of these tumors have been assessed independently in smaller individual cohorts making comparisons difficult. In our study, we analyzed a representative cohort of benign vascular proliferations observed in a clinical routine setting as well as a selection of malignant vascular proliferations. Our cohort of 104 vascular proliferations including hemangiomas, malformations, angiosarcomas and Kaposi’s sarcoma were screened by targeted next-generation sequencing for activating genetic mutations known or assumed to be potentially relevant in vascular proliferations. An association analysis was performed for mutation status and clinico-pathological parameters. Frequent activating hotspot mutations in GNA genes, including GNA14 Q205, GNA11 and GNAQ Q209 were identified in 16 of 64 benign vascular tumors (25%). GNA gene mutations were particularly frequent (52%) in cherry (senile) hemangiomas (13 of 25). In angiosarcomas, activating RAS mutations (HRAS and NRAS) were identified in three samples (16%). No activating GNA or RAS gene mutations were identified in Kaposi’s sarcomas. Our study identifies GNA14 Q205, GNA11 and GNAQ Q209 mutations as being the most common and mutually exclusive mutations in benign hemangiomas. These mutations were not identified in malignant vascular tumors, which could be of potential diagnostic value in distinguishing these entities.

The Emerging Role of Ras Pathway Signaling in Pediatric Cancer.

As genomic sequencing has become more widely available, the high prevalence of Ras pathway mutations in pediatric diseases has begun to emerge. Germline Ras-activating mutations have been known to contribute to cancer predisposition in a group of disorders known as the RASopathies, and now large pediatric sequencing studies have identified frequent somatic Ras pathway alterations across a diverse group of pediatric malignancies. These include glial brain tumors, relapsed high-risk neuroblastoma, embryonal rhabdomyosarcoma, acute myeloid leukemia, and relapsed acute lymphoblastic leukemia, and their prognostic impact is becoming increasingly better understood. Clinically, there has been success in targeting the Ras pathway in pediatric diseases, including the use of MEK inhibitors in plexiform neurofibromas associated with neurofibromatosis type 1 and the use of Ras pathway inhibitors in low-grade gliomas. Given the importance of this pathway in pediatric cancer, it is imperative that future studies strive to better understand the functional significance of these mutations, including their role in tumor growth and treatment resistance and how they can be better targeted to improve outcomes.

RAS-mutated sporadic medullary thyroid cancer: A single-center experience.

6584 Background: Activating RAS mutations are recognized as important drivers in sporadic medullary thyroid cancer (sMTC), with a reported prevalence between 0-43%. However, few studies have looked at correlations between RAS-mutated sMTC and clinicopathologic features. Methods: Patients with sMTC diagnosed between 1992 – 2019 with NGS testing for RET and RAS mutations seen at a tertiary cancer center were retrospectively evaluated. The objective was to analyze demographic and clinical features among patients with RAS-mutated sMTC and to evaluate associations between these features and overall survival (OS). Analyses were performed to correlate patient demographics and pathologic staging with treatment characteristics, disease course, and OS. Results: We identified 42 patients (50% female) with RAS-mutated sMTC out of 218 pts with sMTC. Median age at diagnosis was 50 years (range 24-78 years). 26 (62%) patients had stage IV disease at time of diagnosis. 28 (67%) of patients had HRAS mutations and 14 (33%) had KRAS mutations. HRAS Q61R was the most common HRAS mutation type (n = 19, 45%). Median follow-up time was 64 months (range 23-274 months) during which 11 (26%) patients died. The median OS was 16.2 years, with 5- and 10- year OS of 88% and 73% respectively. Of the 20 (48%) patients who received systemic therapy, 79% had stage IV disease and tended to be older (median age 54). Median time from diagnosis to initiation of systemic therapy was 33 months. Factors associated with worse OS included distant metastases at diagnosis, shorter time interval between diagnosis and treatment, and Ctn/CEA doubling times &lt; 6 months. HRAS Q61R mutations were associated with a better prognosis, with 100% 10-year OS compared with 10-year OS of 39% and 51% (p = 0.02) for other HRAS and KRAS mutations respectively. Conclusions: At a tertiary cancer center, patients with RAS-mutated sMTC had a 10-year OS rate of 73%, with significantly worse OS in patients with HRAS/KRAS mutations other than HRAS Q61R. In comparison, prior studies have reported 10-year OS rates between ~71-90% in sMTC and 10-year OS rates as low as 56% for more aggressive RET M918T sMTC mutations. The findings here are consistent with other studies that have suggested patients with RAS-mutated sMTC are at intermediate risk for aggressive disease, though there are limited data on OS rates in RAS or RAS-/RET- sMTC. Future research comparing outcomes between various RAS mutations and in comparison to RET+ and RAS-/RET- patients is needed, especially as systemic therapy use in RAS-mutated sMTC evolves.

Therapeutic peptides targeting the Ras superfamily

AbstractThe Ras superfamily of small GTPases are master regulators of numerous essential processes within the cell, so that when they malfunction, cancer and many other diseases can result. For example, activating Ras mutations are present in approximately 20% of human cancers. As such, they are key therapeutic targets, yet more than three decades of intensive research efforts have failed to produce effective Ras inhibitors in the clinic. This is, in part, due to their relatively smooth surfaces which are difficult to target through traditional drug discovery methods using small molecules. Peptides offer a solution to this issue as they occupy larger surface areas on their targets and therefore offer exquisite selectivity and affinity. However, their use in the past has been limited to extracellular targets due to delivery issues. Recent advances in peptide macrocyclisation, modifications and delivery methods have ignited increased interest in the use of these highly effective biologics for intracellular targets. This review will cover progress made in the development of peptides targeting small GTPases to treat a wide range of diseases.