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.

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

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.

Comprehensive molecular characterization of a rare case of Philadelphia chromosome-positive acute myeloid leukemia.

The Philadelphia chromosome (Ph) resulting from the t(9;22) translocation generates the oncogenic BCR::ABL1 fusion protein that is most commonly associated with chronic myeloid leukemia (CML) and Ph-positive (Ph+) acute lymphoblastic leukemia (ALL). There are also rare instances of patients (≤1%) with newly diagnosed acute myeloid leukemia (AML) that harbor this translocation (Paietta et al., Leukemia 12: 1881 [1998]; Keung et al., Leuk Res 28: 579 [2004]; Soupir et al., Am J Clin Pathol 127: 642 [2007]). AML with BCR::ABL has only recently been provisionally classified by the World Health Organization as a diagnostically distinct subtype of AML. Discernment from the extremely close differential diagnosis of myeloid blast crisis CML is challenging, largely relying on medical history rather than clinical characteristics (Arber et al., Blood 127: 2391 [2016]). To gain insight into the genomic features underlying the evolution of AML with BCR::ABL, we identified a patient presenting with a high-risk myelodysplastic syndrome that acquired a BCR::ABL alteration after a peripheral blood stem cell transplant. Serial samples were collected and analyzed using whole-exome sequencing, RNA-seq, and ex vivo functional drug screens. Persistent subclones were identified, both at diagnosis and at relapse, including an SF3B1p.Lys700Glu mutation that later cooccurred with an NRASp.Gly12Cys mutation. Functional ex vivo drug screening performed on primary patient cells suggested that combination therapies of ABL1 with RAS or PI3K pathway inhibitors could have augmented the patient's response throughout the course of disease. Together, our findings argue for the importance of genomic profiling and the potential value of ABL1 inhibitor-inclusive combination treatment strategies in patients with this rare disease.

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.

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in JAK2V617F-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.