Samuel B. Luty

TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

The CSF3R T618I mutation causes a lethal neutrophilic neoplasia in mice that is responsive to therapeutic JAK inhibition

We have recently identified targetable mutations in CSF3R (GCSFR) in 60% of chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL-negative) chronic myeloid leukemia (aCML) patients. Here we demonstrate that the most prevalent, activating mutation, CSF3R T618I, is sufficient to drive a lethal myeloproliferative disorder in a murine bone marrow transplantation model. Mice transplanted with CSF3R T618I-expressing hematopoietic cells developed a myeloproliferative disorder characterized by overproduction of granulocytes and granulocytic infiltration of the spleen and liver, which was uniformly fatal. Treatment with the JAK1/2 inhibitor ruxolitinib lowered the white blood count and reduced spleen weight. This demonstrates that activating mutations in CSF3R are sufficient to drive a myeloproliferative disorder resembling aCML and CNL that is sensitive to pharmacologic JAK inhibition. This murine model is an excellent tool for the further study of neutrophilic myeloproliferative neoplasms and implicates the clinical use of JAK inhibitors for this disease.

Ligand independence of the T618I mutation in the colony-stimulating factor 3 receptor (CSF3R) protein results from loss of O-linked glycosylation and increased receptor dimerization.

Background: We recently identified CSF3R mutations in chronic neutrophilic leukemia. The most common mutation, T618I, signals without ligand through an undefined mechanism. Results: CSF3R T618I abrogates an O-glycosylation event and increases receptor dimerization. Conclusion: The constitutive activity of CSF3R T618I may be explained by loss of O-glycosylation. Significance: This study illustrates the role of O-linked glycosylation in protein function and oncogenesis. Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.

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.

Identification and Characterization of Tyrosine Kinase Nonreceptor 2 Mutations in Leukemia through Integration of Kinase Inhibitor Screening and Genomic Analysis

The amount of genomic information about leukemia cells currently far exceeds our overall understanding of the precise genetic events that ultimately drive disease development and progression. Effective implementation of personalized medicine will require tools to distinguish actionable genetic alterations within the complex genetic landscape of leukemia. In this study, we performed kinase inhibitor screens to predict functional gene targets in primary specimens from patients with acute myeloid leukemia and chronic myelomonocytic leukemia. Deep sequencing of the same patient specimens identified genetic alterations that were then integrated with the functionally important targets using the HitWalker algorithm to prioritize the mutant genes that most likely explain the observed drug sensitivity patterns. Through this process, we identified tyrosine kinase nonreceptor 2 (TNK2) point mutations that exhibited oncogenic capacity. Importantly, the integration of functional and genomic data using HitWalker allowed for prioritization of rare oncogenic mutations that may have been missed through genomic analysis alone. These mutations were sensitive to the multikinase inhibitor dasatinib, which antagonizes TNK2 kinase activity, as well as novel TNK2 inhibitors, XMD8-87 and XMD16-5, with greater target specificity. We also identified activating truncation mutations in other tumor types that were sensitive to XMD8-87 and XMD16-5, exemplifying the potential utility of these compounds across tumor types dependent on TNK2. Collectively, our findings highlight a more sensitive approach for identifying actionable genomic lesions that may be infrequently mutated or overlooked and provide a new method for the prioritization of candidate genetic mutations.

Therapeutically Targetable ALK Mutations in Leukemia

Genome sequencing is revealing a vast mutational landscape in leukemia, offering new opportunities for treatment with targeted therapy. Here, we identify two patients with acute myelogenous leukemia and B-cell acute lymphoblastic leukemia whose tumors harbor point mutations in the ALK kinase. The mutations reside in the extracellular domain of ALK and are potently transforming in cytokine-independent cellular assays and primary mouse bone marrow colony formation studies. Strikingly, both mutations conferred sensitivity to ALK kinase inhibitors, including the FDA-approved drug crizotinib. On the basis of our results, we propose that tumors harboring ALK mutations may be therapeutically tractable for personalized treatment of certain aggressive leukemias with ALK inhibitors.

Characterization of the leukemogenic potential of distal cytoplasmic CSF3R truncation and missense mutations

An increasing number of variants of unknown significance are being identified in leukemia patients with the application of deep sequencing and these include CSF3R cytoplasmic mutations. Previous studies have demonstrated oncogenic potential of certain CSF3R truncation mutations prior to internalization motifs. However, the oncogenic potential of truncating the more distal region of CSF3R cytoplasmic domain as well as cytoplasmic missense mutations remains uncharacterized. Here we identified that CSF3R distal cytoplasmic truncation mutations (Q793–Q823) also harbored leukemogenic potential. Mechanistically, these distal cytoplasmic truncation mutations demonstrated markedly decreased receptor degradation, probably owing to loss of the de-phosphorylation domain (residues N818–F836). Furthermore, all truncations prior to Q823 demonstrated increased expression of the higher molecular weight CSF3R band, which is shown to be essential for the receptor surface expression and the oncogenic potential. We further demonstrated that sufficient STAT5 activation is essential for oncogenic potential. In addition, CSF3R K704A demonstrated transforming capacity due to interruption of receptor ubiquitination and degradation. In summary, we have expanded the region of the CSF3R cytoplasmic domain in which truncation or missense mutations exhibit leukemogenic capacity, which will be useful for evaluating the relevance of CSF3R mutations in patients and helpful in defining targeted therapy strategies.

Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia

Dasatinib or TNK2-specific inhibitors may treat PTPN11-mutant leukemia. A ready-to-go treatment for AML and JMML In some acute myeloid and juvenile myelomonocytic leukemias (AML and JMML, respectively), tumor growth is driven by activating mutations in the phosphatase PTPN11. Jenkins et al. found that mutant PTPN11 activity is enhanced by the kinase TNK2. The multikinase inhibitor dasatinib decreased the activity of TNK2, mutant PTPN11, and downstream proliferative pathways in patient cells in culture; decreased their growth in murine bone marrow cocultures; and extended survival in a patient with PTPN11-mutant JMML. Although dasatinib does not block mutant PTPN11 activity entirely, it is clinically approved for the treatment of other leukemias (CML and ALL), suggesting that its use could be extended to AML and JMML to slow disease progression in patients. The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell–based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias.

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.

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

BackgroundLiposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management.MethodsLarge RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial.ResultsScreening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model.ConclusionsTwo large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.