Samantha L. Savage

The Colony-Stimulating Factor 3 Receptor T640N Mutation Is Oncogenic, Sensitive to JAK Inhibition, and Mimics T618I

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. Experimental Design: Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. Results: Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation—despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. Conclusions: Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML. Clin Cancer Res; 22(3); 757–64. ©2015 AACR.

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies

Significance Mononuclear cells obtained from freshly isolated patient samples with various hematologic malignancies were evaluated for sensitivities to combinations of drugs that target specific cell-signaling pathways. The diagnostic, genetic/cytogenetic, and cellular features of the patient samples were correlated with effective drug combinations. For myeloid-derived tumors, such as acute myeloid leukemia, several combinations of targeted agents that include a kinase inhibitor and venetoclax, a selective inhibitor of BCL2, are effective. Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

Super-enhancers promote transcriptional dysregulation in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614-26. ©2017 AACR.

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in JAK2 V617F -positive myeloproliferative neoplasms

The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.

Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

Abstract 2760: CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have recently discovered that a large percentage (59%) of patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML) have mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) (Maxson et al, NEJM 2013). These mutations are either point mutations in the extracellular domain (T618I, T615A) or truncations of the cytoplasmic domain. The most frequent mutation is T618I (aka T595I). The two classes of CSF3R mutations operate through distinct mechanisms that result in differential sensitivity to inhibition of tyrosine kinases downstream of CSF3R. While the mechanism of action of CSF3R truncation mutations has been well characterized-they have increased cell surface expression due to loss of negative regulatory motifs-the way in which the CSF3R point mutations activate the receptor is unclear. Consistent with a previous report, the CSF3R T618I mutation is ligand independent in both mouse bone marrow colony assays and the Ba/F3 pro-B cell line. WT CSF3R consists of two molecular weight species suggestive of differential posttranslational modification. Analysis of CSF3R point mutations T618I and T615A revealed an alteration of banding pattern of these mutations. Since extracellular threonine residues are capable of being O-glycosylated, we employed endoglycosidase digestion to reveal that the larger CSF3R species is O-glycosylated. Furthermore the T618I and T615A mutations reduce O-glycosylation of the receptor. This analysis was confirmed by direct labeling of O-glycosylation using azide-modified galactosamine, which labeled WT CSF3R, but had minimal labeling of CSF3R T618I. Receptor dimerization studies showed increased dimerization of the CSF3R T618I mutant relative to WT. Sequencing of CSF3R from an atypical CML patient samples revealed an additional mutation not previously observed in this disease, T640N. The T640N mutation (aka T617N) is a ligand-independent mutation originally found in a family with hereditary chronic neutrophilia (Plo et al, JEM 2009). Plo et al found that this mutation resides within the transmembrane domain and predicted by molecular modeling that it increased receptor dimerization through interaction of the transmembrane helices. In mouse bone marrow colony assays we confirmed the ligand-independence of the mutation and also found that it had similar potency as T618I in colony and cytokine-independent growth assays. Furthermore, like T618I, the T640N mutation conferred sensitivity to a JAK kinase inhibitor in colony assays. Surprisingly, the T640N mutation had an alteration of banding pattern very similar to T618I, suggesting that it may also have altered glycosylation status, even though the mutation resides within the transmembrane domain. Our studies demonstrate that O-glycosylation is critical for regulation of CSF3R dimerization and that the T618I and T640N mutations can activate the receptor to promote oncogenesis. Citation Format: Julia Maxson, Samuel Luty, Jason MacManiman, Samantha Savage, Melissa Abel, Swaleh Bahamadi, Brian Druker, Jeffrey Tyner. CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2014-2760