Michael R. Rossi

Chronic myeloproliferative neoplasms (MPN) frequently evolve to a blast phase (BP) that is almost uniformly resistant to induction chemotherapy or hypomethylating agents. We explored the functional properties, genomic architecture, and cell of origin of MPN-BP initiating cells (IC) using a serial NSG mouse xenograft transplantation model. Transplantation of peripheral blood mononuclear cells (MNC) from 7 of 18 patients resulted in a high degree of leukemic cell chimerism and recreated clinical characteristics of human MPN-BP. The function of MPN-BP ICs was not dependent on the presence of JAK2V617F , a driver mutation associated with the initial underlying MPN. By contrast, multiple MPN-BP IC subclones coexisted within MPN-BP MNCs characterized by different myeloid malignancy gene mutations and cytogenetic abnormalities. MPN-BP ICs in 4 patients exhibited extensive proliferative and self-renewal capacity, as demonstrated by their ability to recapitulate human MPN-BP in serial recipients. These MPN-BP IC subclones underwent extensive continuous clonal competition within individual xenografts and across multiple generations, and their subclonal dynamics were consistent with functional evolution of MPN-BP IC. Finally, we show that MPN-BP ICs originate from not only phenotypically identified hematopoietic stem cells, but also lymphoid-myeloid progenitor cells, which were each characterized by differences in MPN-BP initiating activity and self-renewal capacity.

Historically, high rates of actionable driver mutations have been reported in never-smokers with lung adenocarcinoma (ADC). In the era of modern, comprehensive cancer mutation sequencing, this relationship necessitates a more detailed analysis. All Mount Sinai patients between January 1, 2015, and June 1, 2020, with a diagnosis of ADC of any stage with known smoking status who received genomic testing were included. Most patients were analyzed using the Sema4 hotspot panel or the Oncomine Comprehensive Assay version 3 next-generation sequencing (NGS) panel conducted at Sema4. Patients were considered fully genotyped if they were comprehensively analyzed for alterations in EGFR, KRAS, MET, ALK, RET, ROS1, BRAF, NTRK1-3, and ERBB2, otherwise they were considered partially genotyped. Two hundred and thirty-six never-smokers and 671 smokers met the above criteria. Of the never-smokers, 201 (85%) had a driver mutation with 167 (71%) considered actionable (ie, those with US Food and Drug Administration-approved agents). Among smokers, 439 (65%) had an identified driver mutation with 258 (38%) actionable (P < .0001). When comprehensively sequenced, 95% (70/74) of never-smokers had a driver mutation with 78% (58/74) actionable; whereas, for smokers, 75% (135/180) had a driver with only 47% (74/180) actionable (P < .0001). Within mutations groups, EGFR G719X and KRAS G12Cs were more common to smokers. For stage IV patients harboring EGFR-mutant tumors treated with EGFR-directed therapies, never-smokers had significantly improved OS compared to smokers (hazard ratio = 2.71; P = .025). In multivariable analysis, Asian ancestry and female sex remained significant predictors of (1) OS in stage IV patients and (2) likelihood of harboring a receptor of fusion-based driver. Comprehensive NGS revealed driver alterations in 95% of never-smokers, with the majority having an associated therapy available. All efforts should be exhausted to identify or rule out the presence of an actionable driver mutation in all metastatic lung ADC.

Activation of the P53 pathway through inhibition of MDM2 using nutlins has shown clinical promise in the treatment of solid tumors and hematologic malignancies. There is concern, however, that nutlin therapy might stimulate the emergence or expansion of TP53-mutated subclones. We recently published the results of a phase 1 trial of idasanutlin in patients with polycythemia vera (PV) that revealed tolerability and clinical activity. Here, we present data indicating that idasanutlin therapy is associated with expansion of TP53 mutant subclones. End-of-study sequencing of patients found that 5 patients in this trial harbored 12 TP53 mutations; however, only 1 patient had been previously identified as having a TP53 mutation at baseline. To identify the origin of these mutations, further analysis of raw sequencing data of baseline samples was performed and revealed that a subset of these mutations was present at baseline and expanded during treatment with idasanutlin. Follow-up samples were obtained from 4 of 5 patients in this cohort, and we observed that after cessation of idasanutlin, the variant allele frequency (VAF) of 8 of 9 TP53 mutations decreased. Furthermore, disease progression to myelofibrosis or myeloproliferative neoplasm blast phase was not observed in any of these patients after 19- to 32-month observation. These data suggest that idasanutlin treatment may promote transient TP53 mutant clonal expansion. A larger study geared toward high-resolution detection of low VAF mutations is required to explore whether patients acquire de novo TP53 mutations after idasanutlin therapy.

Allele-specific copy number analysis of tumors (ASCAT) assesses copy number variations (CNV) while accounting for aberrant cell fraction and tumor ploidy. We evaluated if ASCAT-assessed CNV are associated with survival outcomes in 56 patients with WHO grade IV gliomas. Tumor data analyzed by Affymetrix OncoScan FFPE Assay yielded the log ratio (R) and B-allele frequency (BAF). Input into ASCAT quantified CNV using the segmentation function to measure copy number inflection points throughout the genome. Quantified CNV was reported as log R and BAF segment counts. Results were confirmed on The Cancer Genome Atlas (TCGA) glioblastoma dataset. 25 (44.6%) patients had MGMT hyper-methylated tumors, 6 (10.7%) were IDH1 mutated. Median follow-up was 36.4 months. Higher log R segment counts were associate with longer progression-free survival (PFS) [hazard ratio (HR) 0.32, p < 0.001], and overall survival (OS) [HR 0.45, p = 0.01], and was an independent predictor of PFS and OS on multivariable analysis. Higher BAF segment counts were linked to longer PFS (HR 0.49, p = 0.022) and OS (HR 0.49, p = 0.052). In the TCGA confirmation cohort, longer 12-month OS was seen in patients with higher BAF segment counts (62.3% vs. 51.9%, p = 0.0129) and higher log R (63.6% vs. 55.2%, p = 0.0696). Genomic CNV may be a novel prognostic biomarker for WHO grade IV glioma patient outcomes.

Amino acid metabolism is involved in diverse cellular functions, including cell survival and growth; however, it remains unclear how it regulates normal haematopoiesis versus leukaemogenesis. Here, we report that knockout of solute carrier family 1 member 5 (Slc1a5/ASCT2), a transporter of neutral amino acids, especially glutamine, results in mild-to-moderate defects in bone marrow and mature blood cell development under steady-state conditions. In contrast, constitutive or induced deletion of Slc1a5 decreases leukaemia initiation and maintenance driven by oncogene MLL-AF9 or phosphatase and tensin homologue (Pten) deficiency. Survival of leukaemic mice is prolonged following Slc1a5 deletion, and pharmacological inhibition of ASCT2 also decreases leukaemia development and progression in xenograft models of human acute myeloid leukaemia. Mechanistically, loss of ASCT2 generates a global effect on cellular metabolism, disrupts leucine (Leu) influx and mechanistic target of rapamycin (mTOR) signalling, and induces apoptosis in leukaemic cells. Given the substantial difference in reliance on ASCT2-mediated amino acid metabolism between normal and malignant blood cells, this in vivo study suggests ASCT2 as a promising therapeutic target for the treatment of leukaemia.

BCL2 family members such as MCL1, BCLX_L, and BCL2 are critical for cancer cell survival and therefore represent promising therapeutic targets. Both B cells and CLL cells depend primarily on BCL-2 and are thus sensitive to the BCL2 specific inhibitor venetoclax, while plasma cells and multiple myeloma typically depend on Mcl-1 and would therefore be resistant to venetoclax. However, a subset of myeloma is venetoclax sensitive based on recent in vitro and clinical trial data. In preliminary results from a phase I trial of venetoclax in multiple myeloma, 40% of patients positive for t(11;14) had objective responses, while only 6% of t(11;14) negative patients responded. We have made similar observations with in vitro testing of 30 freshly isolated myeloma patient samples, identifying both non-t(11;14) samples sensitive to venetoclax as well as resistant t(11;14) positive samples. Together, these results suggest not only that a subset of multiple myeloma is co-dependent on BCL2 but also that t(11;14) is neither necessary nor sufficient for responding to venetoclax. We therefore set out to identify other factors that may predict BCL2 dependence in multiple myeloma. Previous studies of t(11;14) myeloma have noted increased expression of CD20, CD23, CD79a, and PAX5 which are typically associated with B cells prior to their differentiation into plasma cells. Based on these observations we hypothesized that venetoclax sensitivity in myeloma may be associated with the retention of B cell properties including BCL2 dependence. We probed an online expression database of myeloma cell lines for non-t(11;14) cell lines expressing CD20 and identified two cell lines, OCI-My5 and PCM6, both of which we found to have an IC50 of approximately 50nM when treated with venetoclax. We went on to characterize a panel of 13 cell lines. In addition to OCI-My5 and PCM6, 4 other cell lines were sensitive to venetoclax, all positive for t(11;14). Of the 7 venetoclax resistant cell lines, 2 were t(11;14) positive. Protein levels of MCL1, BCLX_L, and BCL2 were comparable among the 13 lines and therefore anti-apoptotic expression is unlikely to be responsible for venetoclax sensitivity. Consistent with our previous co-immunoprecipitation studies, more of the pro-apoptotic BIM was bound to BCL2 in venetoclax sensitive lines compared to resistant lines. In the absence of differences in BCL2 family expression, we next sought to identify other B cell related features correlating with venetoclax sensitivity. We used RNAseq data from our 13 cell lines to compare the expression of 100 genes previously reported to be differentially expressed between normal B cells and plasma cells. Interestingly, unsupervised clustering revealed a group of venetoclax sensitive cells enriched for other B cell associated genes. GSEA revealed enrichment of genes associated with immune system activation at a p < 0.001. We also analyzed the differential expression of genes between our sensitive and resistant lines and again identified overexpression of B cell related genes such as CD20, CD79A, STAT5A, and RASGRP2 in venetoclax sensitive lines, though no single marker was present in all of the venetoclax sensitive lines. We examined the expression of CD20, CD79a, and CD79b in the CoMMpass data set (IA8) as well and found that they were not co-expressed in most patients, again suggesting that no single marker is likely to be predictive. Finally, we created a gene signature from the top differentially expressed genes to predict sensitivity or resistance to venetoclax and used this signature to evaluate a database of 68 myeloma cell lines. One of the top hits predicted to be sensitive by our gene signature is the t(11;14) negative line MOLP2, and indeed this cell line was recently reported to be highly responsive to venetoclax. In conclusion, B cell markers and our gene signature correlate with BCL2 dependence and venetoclax sensitivity independent of t(11;14).

Copy number abnormalities (CNAs) in tumor cells are presumed to affect expression levels of genes located in region of abnormality. To investigate this relationship we have surveyed the losses, gains and amplifications in 30 glioblastomas using array comparative genome hybridization and compared these data with gene expression changes in the same tumors using the Affymetrix U133Plus2.0 oligonucleotide arrays. The two datasets were overlaid using our in‐house overlay tool which highlights concordance between CNAs and expression level changes for the same tumors. In this survey we have highlighted genes frequently overexpressed in amplified regions on chromosomes 1, 4, 11, and 12 and have identified novel amplicons on these chromosomes. Deletions of specific regions on chromosomes 9, 10, 11, 14, and 15 have also been correlated with reduced gene expression in the regions of minimal overlap. In addition we describe a novel approach for comparing gene expression levels between tumors based on the presence or absence of chromosome CNAs. This genome wide screen provides an efficient and comprehensive survey of genes which potentially serve as the drivers for the CNAs in GBM.