Tyrosine kinase inhibitor (TKI) therapy for human cancers is not curative, with relapse due to the continuing presence of tumor cells, referred to as minimal residual disease (MRD) cells. MRD stem or progenitor cells survival in the absence of oncogenic kinase signaling, a phenomenon referred to as intrinsic resistance, depends on diverse growth factors. Here, we report that oncogenic kinase and growth factor signaling converge to induce the expression of the signaling proteins c-Fos and Dusp1. Genetic deletion of c-Fos and Dusp1 suppressed tumor growth in a BCR-ABL-induced mouse model of chronic myeloid leukemia (CML). Pharmacological inhibition of c-Fos, Dusp1 and BCR-ABL eradicated MRD in multiple in vivo models, as well as in primary CML patient xenotransplanted mice. Growth factor signaling also conferred TKI resistance and induced c-FOS and DUSP1 expression in tumor cells modeling other types of kinase-driven leukemias. Our data demonstrate that c-Fos and Dusp1 expression levels determine the threshold of TKI efficacy, such that growth factor-induced expression of c-Fos and Dusp1 confers intrinsic resistance to TKI therapy in a wide-ranging set of leukemias, and may represent a unifying Achilles heel of kinase-driven cancers.

Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.

Both membrane-proximal and truncation mutations in CSF3R have recently been reported to drive the onset of chronic neutrophilic leukemia (CNL). Here we show that although truncation mutation alone can not induce leukemia, both proximal and compound mutations (proximal and truncation mutations on same allele) are leukemogenic with a disease latency of 90 and 23 days, respectively. Comparative whole-genome expression profiling and biochemical experiments revealed that induced expression of Mapk adaptor protein Ksr1 and enhanced Mapk signaling are crucial to leukemogenesis by CSF3R proximal and compound mutants. Moreover, inhibition of Mek1/2 by trametinib alone is sufficient to suppress leukemia induced by both CSF3R proximal and ruxolitinib-resistant compound mutations. Together, these findings elucidate a Mapk-dependent mechanism of CSF3R-induced pathogenesis, and they establish the rationale for clinical evaluation of MEK1/2 inhibition in CNL.

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Essential thrombocythemia (ET) is a myeloproliferative neoplasm that is characterized by pathological overproduction of platelets with an increased risk of thromboembolic events, progression to myelofibrosis and leukemic transformation. Mutations of the Janus Kinase 2 (JAK2) gene, most commonly JAK2V617F, are known driving factors of ET pathogenesis. However, the molecular processes involved in JAK2 activation and regulation and the specific mechanism(s) by which JAK2 mutations disrupt hematopoiesis remain unclear. Most mutations identified in hereditary ET involve the thrombopoietin or thrombopoietin receptor gene; only 4 germline JAK2 mutations have been reported, all of which are inherited in an autosomal dominant pattern. We report the first case in which 2 novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, were found in the compound heterozygous state in 2 siblings with ET. Family members who carry either one of these variants have normal blood counts. The purpose of this study was to demonstrate causality and to explore the mechanism(s) by which these JAK2 mutations, in compound heterozygous state, result in thrombopoietic dysregulation. To study signaling in the JAK2-signal transducer and activator of transcription (STAT) pathway, wild-type (WT) and mutated forms of murine JAK2 were transduced into murine pro-B cells (Ba/F3) stably expressing the thrombopoietin receptor (MPL), Ba/F3-MPL cells. Immunoblotting analysis was performed on Ba/F3-MPL cells that were starved of cytokines overnight and then treated with 10 ng/mL thrombopoietin (TPO) or vehicle for 5 minutes. At baseline (under starved conditions), JAK2L815P + JAK2V1123G Ba/F3-MPL cells demonstrated increased STAT3 and STAT5 phosphorylation compared to WT JAK2 Ba/F3-MPL cells. However, JAK2 double mutant Ba/F3-MPL cells had less JAK2, STAT1 and STAT3 phosphorylation in comparison to JAK2V617F Ba/F3-MPL cells at baseline. After TPO stimulation, both JAK2 double mutant and JAK2V617F Ba/F3-MPL cells showed increased phosphorylated JAK2, STAT1 and STAT3 compared to WT JAK2 Ba/F3-MPL cells. To investigate JAK2-STAT signaling in the patients' cells, immunoblotting analyses of platelets and megakaryocytes grown in vitro from patients' CD34 cells were performed. Our patients' platelets demonstrated increased JAK2, STAT1, STAT3 and STAT5 phosphorylation compared to platelets from a normal control but less JAK2, STAT1 and STAT3 phosphorylation compared to the platelets from a pediatric ET patient with a known JAK2V617F mutation, a similar signaling pattern to that seen in JAK2 double mutant Ba/F3-MPL cells. Similarly, in vitro megakaryocytes from the patients' CD34 cells showed increased STAT5 activation compared to those from normal volunteers in the unstimulated state. To assess cell proliferative capability, IL-3 dependent Ba/F3 cells were starved of cytokines and viable cells were counted every 24 hours for a total of 72 hours. The JAK2V617F and JAK2 double mutant Ba/F3-MPL cells easily proliferated without cytokines and had a large fold-increase at 24, 48 and 72 hours compared to 0 hours. The JAK2L815P + JAK2V1123G Ba/F3-MPL cells had a significantly higher proliferation rate compared to JAK2V617F and WT JAK2 Ba/F3-MPL cells. As expected, the number of viable parental Ba/F3 cells declined over time in the absence of cytokines. To evaluate response to ruxolitinib, a JAK1 and JAK2 inhibitor, IL-3 independent Ba/F3-MPL cells were grown under starved conditions in the presence of 0 to 1 mM ruxolitinib. After 48 hours, the number of viable Ba/F3-MPL cells was measured as a percentage of the dimethylsulfoxide (DMSO) control. The 50% inhibitory concentration (IC50) value was higher in JAK2L815P + JAK2V1123G Ba/F3-MPL cells (147+/-20 nM) compared to JAK2V617F Ba/F3-MPL cells (93+/-1 nM). In the compound heterozygous state, the novel germline JAK2 mutations, JAK2L815P and JAK2V1123G, appear to function cooperatively to increase cell proliferation through activation of the JAK2-STAT pathway. This finding implicates that within the JAK2 dimer in the MPL-JAK2-STAT pathway, the monomer-monomer interactions are important in JAK2 activation and regulation. Additional in vitro and in vivo studies are currently underway to elucidate the exact mechanism(s) of thrombopoietic dysregulation resulting in the siblings' ET and to gain further insight into JAK2 activation and regulation. Disclosures Raj: Novartis: Speakers Bureau. Kalfa:Baxter/Baxalta/Shire: Research Funding.

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Disclosures No relevant conflicts of interest to declare.

Disclosures No relevant conflicts of interest to declare.