SHP2 inhibition displays efficacy as a monotherapy and in combination with JAK2 inhibition in preclinical models of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocytosis, and primary myelofibrosis, are clonal hematopoietic neoplasms driven by mutationally activated signaling by the JAK2 tyrosine kinase. Although JAK2 inhibitors can improve MPN patients' quality of life, they do not induce complete remission as disease-driving cells persistently survive therapy. ERK activation has been highlighted as contributing to JAK2 inhibitor persistent cell survival. As ERK is a component of signaling by activated RAS proteins and by JAK2 activation, we sought to inhibit RAS activation to enhance responses to JAK2 inhibition in preclinical MPN models. We found the SHP2 inhibitor RMC-4550 significantly enhanced growth inhibition of MPN cell lines in combination with the JAK2 inhibitor ruxolitinib, effectively preventing ruxolitinib persistent growth, and the growth and viability of established ruxolitinib persistent cells remained sensitive to SHP2 inhibition. Both SHP2 and JAK2 inhibition diminished cellular RAS-GTP levels, and their concomitant inhibition enhanced ERK inactivation and increased apoptosis. Inhibition of SHP2 inhibited the neoplastic growth of MPN patient hematopoietic progenitor cells and exhibited synergy with ruxolitinib. RMC-4550 antagonized MPN phenotypes and increased survival of an MPN mouse model driven by MPL-W515L. The combination of RMC-4550 and ruxolitinib, which was safe and tolerated in healthy mice, further inhibited disease compared to ruxolitinib monotherapy, including extending survival. Given SHP2 inhibitors are undergoing clinical evaluation in patients with solid tumors, our preclinical findings suggest that SHP2 is a candidate therapeutic target with potential for rapid translation to clinical assessment to improve current targeted therapies for MPN patients.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Lowering Sample Requirements to Study Tyrosine Kinase Signaling Using Phosphoproteomics with the TMT Calibrator Approach.

Analysis of tyrosine kinase signaling is critical for the development of targeted cancer therapy. Currently, immunoprecipitation of phosphotyrosine (pY) peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to profile tyrosine kinase substrates. A typical protocol requests 10 mg of total protein from ≈108 cells or 50-100 mg of tissue. Large sample requirements can be cost prohibitive or not feasible for certain experiments. Sample multiplexing using chemical labeling reduces the protein amount required for each sample, and newer approaches use a material-rich reference channel as a calibrator to trigger detection and quantification for smaller samples. Here, it is demonstrated that the tandem mass tag (TMT) calibrator approach reduces the sample input for pY profiling tenfold (to ≈1 mg total protein per sample from 107 cells grown in one plate), while maintaining the depth of pY proteome sampling and the biological content of the experiment. Data are available through PRIDE (PXD019764 for label-free and PXD018952 for TMT). This strategy opens more opportunities for pY profiling of large sample cohorts and samples with limited protein quantity such as immune cells, xenograft models, and human tumors.

Porphyrin overdrive rewires cancer cell metabolism.

All cancer cells reprogram metabolism to support aberrant growth. Here, we report that cancer cells employ and depend on imbalanced and dynamic heme metabolic pathways, to accumulate heme intermediates, that is, porphyrins. We coined this essential metabolic rewiring "porphyrin overdrive" and determined that it is cancer-essential and cancer-specific. Among the major drivers are genes encoding mid-step enzymes governing the production of heme intermediates. CRISPR/Cas9 editing to engineer leukemia cell lines with impaired heme biosynthetic steps confirmed our whole-genome data analyses that porphyrin overdrive is linked to oncogenic states and cellular differentiation. Although porphyrin overdrive is absent in differentiated cells or somatic stem cells, it is present in patient-derived tumor progenitor cells, demonstrated by single-cell RNAseq, and in early embryogenesis. In conclusion, we identified a dependence of cancer cells on non-homeostatic heme metabolism, and we targeted this cancer metabolic vulnerability with a novel "bait-and-kill" strategy to eradicate malignant cells.

JAK2-V617F-mediated signalling is dependent on lipid rafts and statins inhibit JAK2-V617F-dependent cell growth.

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs.

Targeting BET Proteins Downregulates miR-33a To Promote Synergy with PIM Inhibitors in CMML.

PURPOSE: Preclinical studies in myeloid neoplasms have demonstrated efficacy of bromodomain and extra-terminal protein inhibitors (BETi). However, BETi demonstrates poor single-agent activity in clinical trials. Several studies suggest that combination with other anticancer inhibitors may enhance the efficacy of BETi. EXPERIMENTAL DESIGN: To nominate BETi combination therapies for myeloid neoplasms, we used a chemical screen with therapies currently in clinical cancer development and validated this screen using a panel of myeloid cell line, heterotopic cell line models, and patient-derived xenograft models of disease. We used standard protein and RNA assays to determine the mechanism responsible for synergy in our disease models. RESULTS: We identified PIM inhibitors (PIMi) as therapeutically synergistic with BETi in myeloid leukemia models. Mechanistically, we show that PIM kinase is increased after BETi treatment, and that PIM kinase upregulation is sufficient to induce persistence to BETi and sensitize cells to PIMi. Furthermore, we demonstrate that miR-33a downregulation is the underlying mechanism driving PIM1 upregulation. We also show that GM-CSF hypersensitivity, a hallmark of chronic myelomonocytic leukemia (CMML), represents a molecular signature for sensitivity to combination therapy. CONCLUSIONS: Inhibition of PIM kinases is a potential novel strategy for overcoming BETi persistence in myeloid neoplasms. Our data support further clinical investigation of this combination.

Mutations in the transmembrane and juxtamembrane domains enhance IL27R transforming activity.

Cytokines and their receptors regulate haemopoiesis by controlling cellular growth, survival and differentiation. Thus it is not surprising that mutations of cytokine receptors contribute to the formation of haemopoietic disorders, including cancer. We recently identified transforming properties of IL27R, the ligand-binding component of the receptor for interleukin-27. Although wild-type IL27R exhibits transforming properties in haemopoietic cells, in the present study we set out to determine if the transforming activity of IL27R could be enhanced by mutation. We identified three mutations of IL27R that enhance its transforming activity. One of these mutations is a phenylalanine to cysteine mutation at residue 523 (F523C) in the transmembrane domain of the receptor. The two other mutations identified involve deletions of amino acids in the cytoplasmic juxtamembrane region of the receptor. Expression of each of these mutant IL27R proteins led to rapid cytokine-independent transformation in haemopoietic cells. Moreover, the rate of transformation induced by these mutants was significantly greater than that induced by wild-type IL27R. Expression of these IL27R mutants also induced enhanced activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling compared with wild-type. An activating deletion mutation of IL27R enhanced homodimerization of the receptor by a mechanism that may involve disulfide bonding. These transforming IL27R mutants displayed equal or greater transforming activity than bona fide haemopoietic oncogenes such as BCR-ABL (breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue) and JAK2-V617F. Since IL27R is expressed on haemopoietic stem cells, lymphoid cells and myeloid cells, including acute myeloid leukaemia blast cells, mutation of this receptor has the potential to contribute to a variety of haemopoietic neoplasms.

JAK2 inhibitor persistence in MPN: uncovering a central role of ERK activation.

The Philadelphia chromosome negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are driven by hyper activation of the JAK2 tyrosine kinase, the result of mutations in three MPN driving genes: JAK2, MPL, and CALR. While the anti-inflammatory effects of JAK2 inhibitors can provide improved quality of life for many MPN patients, the upfront and persistent survival of disease-driving cells in MPN patients undergoing JAK2 inhibitor therapy thwarts potential for remission. Early studies indicated JAK2 inhibitor therapy induces heterodimeric complex formation of JAK2 with other JAK family members leading to sustained JAK2-dependent signaling. Recent work has described novel cell intrinsic details as well as cell extrinsic mechanisms that may contribute to why JAK2 inhibition may be ineffective at targeting MPN driving cells. Diverse experimental strategies aimed at uncovering mechanistic details that contribute to JAK2 inhibitor persistence have each highlighted the role of MEK/ERK activation. These approaches include, among others, phosphoproteomic analyses of JAK2 signaling as well as detailed assessment of JAK2 inhibition in mouse models of MPN. In this focused review, we highlight these and other studies that collectively suggest targeting MEK/ERK in combination with JAK2 inhibition has the potential to improve the efficacy of JAK2 inhibitors in MPN patients. As MPN patients patiently wait for improved therapies, such studies should further strengthen optimism that pre-clinical research is continuing to uncover mechanistic insights regarding the ineffectiveness of JAK2 inhibitors, which may lead to development of improved therapeutic strategies.

Activation of JAK2-V617F by components of heterodimeric cytokine receptors.

The JAK2-V617F mutation is an important etiologic factor for the development of myeloproliferative neoplasms. The mechanism by which this mutated tyrosine kinase initiates deregulated signals in cells is not completely understood. It is believed that JAK2-V617F requires interactions with homodimeric cytokine receptors to elicit its transforming signal. In this study, we demonstrate that components of heterodimeric cytokine receptors can also activate JAK2-V617F. Expression of IL27Ra, a heterodimeric receptor component, enhanced the activation of JAK2-V617F and subsequent downstream signaling to activation of STAT5 and ERK. In addition, expression of components of the interleukin-3 receptor, IL3Ra and the common beta chain, activated JAK2-V617F as well as STAT5 and ERK. Importantly, expression of IL27Ra functionally replaced the requirement of a homodimeric cytokine receptor to promote the activation and transforming activity of JAK2-V617F in BaF3 cells. Tyrosine phosphorylation of IL27Ra was not required to induce activation of JAK2-V617F or STAT5, or to enhance the transforming activity of JAK2-V617F. Expression of IL3Ra or the common beta chain in BaF3 cells also enhanced the ability of JAK2-V617F to transform these hematopoietic cells. However, the heterodimeric receptor component IL12RB1 did not enhance the activation or transforming signals of JAK2-V617F in BaF3 cells. IL27Ra also activated the K539L and R683G JAK2 mutants. Together our data demonstrate that in addition to homodimeric receptors, some heterodimeric receptor components can support the activation and transforming signals of JAK2-V617F and other JAK2 mutants. Therefore, heterodimeric receptors may play unappreciated roles in JAK2 activation in the development of hematopoietic diseases including myeloproliferative neoplasms.

Structure-function correlation of G6, a novel small molecule inhibitor of Jak2: indispensability of the stilbenoid core.

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.

Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism.

Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2). Once activated, Rac stimulates signalling pathways that regulate actin organization, gene expression and cellular proliferation. Rac also functions downstream of the Ras oncoprotein in pathways that stimulate membrane ruffling, growth transformation, activation of the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase, activation of the NF-kappa B transcription factor and promotion of cell survival. Although recent studies support phosphatidylinositol 3-OH kinase (PI(3)K)-dependent mechanisms through which Ras might activate Rac (refs 9,10), the precise mechanism remains to be determined. Here we demonstrate that Tiam1, a Rac-specific GEF, preferentially associates with activated GTP-bound Ras through a Ras-binding domain. Furthermore, activated Ras and Tiam1 cooperate to cause synergistic formation of Rac-GTP in a PI(3)K-independent manner. Thus, Tiam1 can function as an effector that directly mediates Ras activation of Rac.

Transforming JAK1 mutations exhibit differential signalling, FERM domain requirements and growth responses to interferon-γ.

Recent work has highlighted roles for JAK (Janus kinase) family members in haemopoietic diseases. Although sequencing efforts have uncovered transforming JAK1 mutations in acute leukaemia, they have also identified non-transforming JAK1 mutations. Thus with limited knowledge of the mechanisms of JAK1 activation by mutation, sequencing may not readily identify transforming mutations. Therefore we sought to further understand the repertoire of transforming mutations of JAK1. We identified seven randomly generated transforming JAK1 mutations, including V658L and a deletion of amino acids 629-630 in the pseudokinase domain, as well as L910P, F938S, P960S, K1026E and Y1035C within the kinase domain. These mutations led to differential signalling activation, but exhibited similar transforming abilities, in BaF3 cells. Interestingly, these properties did not always correlate with JAK1 activation-loop phosphorylation. We also identified a JAK1 mutant that did not require a functional FERM (4.1/ezrin/radixin/moesin) domain for transformation. Although we isolated a mutation of JAK1 at residue Val658, which is found mutated in acute leukaemia patients, most of the mutations we identified are within the kinase domain and have yet to be identified in patients. Interestingly, compared with cells expressing JAK1-V658F, cells expressing these mutants had higher STAT1 (signal transducer and activator of transcription 1) phosphorylation and were more sensitive to interferon-γ-mediated growth inhibition. The differential STAT1 activation and interferon-sensitivity of JAK1 mutants may contribute to the determination of which specific JAK1 mutations ultimately contribute to disease and thus are identified in patients. Our characterization of these novel mutations contributes to a better understanding of mutational activation of JAK1.

Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof.

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I cytokine receptor.

From a patient with acute myeloid leukemia (AML), we have identified IL-27Ra (also known as TCCR and WSX1) as a gene whose expression can induce the transformation of hematopoietic cells. IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the ligand binding component of the receptor for IL-27 and functions with the glycoprotein 130 (gp130) coreceptor to induce signal transduction in response to IL-27. We show that IL-27R is expressed on the cell surface of the leukemic cells of AML patients. 32D myeloid cells transformed by IL-27R contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins induces cell cycle arrest and apoptosis in these cells, suggesting the transforming properties of IL-27R depend on the activity of JAK family members. IL-27R also transforms BaF3 cells to cytokine independence. Because BaF3 cells lack expression of gp130, this finding suggests that IL-27R-mediated transformation of hematopoietic cells is gp130-independent. Finally, we show that IL-27R can functionally replace a homodimeric type I cytokine receptor in the activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that IL-27R possesses hematopoietic cell-transforming properties and suggest that, analogous to homodimeric type I cytokine receptors, single-chain components of heterodimeric receptors can also enhance the activation of JAK2-V617F. Therefore, such receptors may play unappreciated roles in MPDs.

Finding a Jill for JAK: Assessing Past, Present, and Future JAK Inhibitor Combination Approaches in Myelofibrosis.

Myelofibrosis (MF) is a myeloproliferative neoplasm hallmarked by the upregulation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway with associated extramedullary hematopoiesis and a high burden of disease-related symptoms. While JAK inhibitor therapy is central to the management of MF, it is not without limitations. In an effort to improve treatment for MF patients, there have been significant efforts to identify combination strategies that build upon the substantial benefits of JAK inhibition. Early efforts to combine agents with additive therapeutic profiles have given way to rationally designed combinations hoping to demonstrate clinical synergism and modify the underlying disease. In this article, we review the preclinical basis and existing clinical data for JAK inhibitor combination strategies while highlighting emerging strategies of particular interest.

Metabolic Vulnerabilities and Epigenetic Dysregulation in Myeloproliferative Neoplasms.

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are associated with clonal myelopoiesis, elevated risk of death due to thrombotic complications, and transformation to acute myeloid leukemia (AML). JAK2 inhibitors improve the quality of life for MPN patients, but these approved therapeutics do not readily reduce the natural course of disease or antagonize the neoplastic clone. An understanding of the molecular and cellular changes requisite for MPN development and progression are needed to develop improved therapies. Recently, murine MPN models were demonstrated to exhibit metabolic vulnerabilities due to a high dependence on glucose. Neoplastic hematopoietic progenitor cells in these mice express elevated levels of glycolytic enzymes and exhibit enhanced levels of glycolysis and oxidative phosphorylation, and the disease phenotype of these MPN model mice is antagonized by glycolytic inhibition. While all MPN-driving mutations lead to aberrant JAK2 activation, these mutations often co-exist with mutations in genes that encode epigenetic regulators, including loss of function mutations known to enhance MPN progression. In this perspective we discuss how altered activity of epigenetic regulators (e.g., methylation and acetylation) in MPN-driving stem and progenitor cells may alter cellular metabolism and contribute to the MPN phenotype and progression of disease. Specific metabolic changes associated with epigenetic deregulation may identify patient populations that exhibit specific metabolic vulnerabilities that are absent in normal hematopoietic cells, and thus provide a potential basis for the development of more effective personalized therapeutic approaches.

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis.

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.

The pan-PIM inhibitor INCB053914 displays potent synergy in combination with ruxolitinib in models of MPN.

Aberrant JAK2 tyrosine kinase signaling drives the development of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. However, JAK2 kinase inhibitors have failed to significantly reduce allele burden in MPN patients, underscoring the need for improved therapeutic strategies. Members of the PIM family of serine/threonine kinases promote cellular proliferation by regulating a variety of cellular processes, including protein synthesis and the balance of signaling that regulates apoptosis. Overexpression of PIM family members is oncogenic, exemplified by their ability to induce lymphomas in collaboration with c-Myc. Thus, PIM kinases are potential therapeutic targets for several malignancies such as solid tumors and blood cancers. We and others have shown that PIM inhibitors augment the efficacy of JAK2 inhibitors by using in vitro models of MPNs. Here we report that the recently developed pan-PIM inhibitor INCB053914 augments the efficacy of the US Food and Drug Administration-approved JAK1/2 inhibitor ruxolitinib in both in vitro and in vivo MPN models. INCB053914 synergizes with ruxolitinib to inhibit cell growth in JAK2-driven MPN models and induce apoptosis. Significantly, low nanomolar INCB053914 enhances the efficacy of ruxolitinib to inhibit the neoplastic growth of primary MPN patient cells, and INCB053914 antagonizes ruxolitinib persistent myeloproliferation in vivo. These findings support the notion that INCB053914, which is currently in clinical trials in patients with advanced hematologic malignancies, in combination with ruxolitinib may be effective in MPN patients, and they support the clinical testing of this combination in MPN patients.

Characterization of epidermal growth factor receptor (EGFR) P848L, an unusual EGFR variant present in lung cancer patients, in a murine Ba/F3 model.

Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) benefit from treatments targeting tyrosine kinase inhibitors (TKIs). However, both intrinsic and acquired resistance of tumors to TKIs are common, and EGFR variants have been identified that are resistant to multiple TKIs. In the present study, we characterized selected EGFR variants previously observed in lung cancer patients and expressed in a murine bone marrow pro-B Ba/F3 cell model. Among these EGFR variants, we report that an exon 20 deletion/insertion mutation S768insVGH is resistant to erlotinib (a first-generation TKI), but sensitive to osimertinib (a third-generation TKI). We also characterized a rare exon 21 germline variant, EGFR P848L, which transformed Ba/F3 cells and conferred resistance to multiple EGFR-targeting TKIs. Our analysis revealed that P848L (a) does not bind erlotinib; (b) is turned over less rapidly than L858R (a common tumor-derived EGFR mutation); (c) is not autophosphorylated at Tyr 1045 [the major docking site for Cbl proto-oncogene (c-Cbl) binding]; and (d) does not bind c-Cbl. Using viability assays including 300 clinically relevant targeted compounds, we observed that Ba/F3 cells transduced with EGFR P848L, S768insVGH, or L858R have very different drug-sensitivity profiles. In particular, EGFR P848L, but not L858R or S768insVGH, was sensitive to multiple Janus kinase 1/2 inhibitors. In contrast, cells driven by L858R, but not by P848L, were sensitive to multikinase MAPK/extracellular-signal-regulated kinase (ERK) kinase and ERK inhibitors including EGFR-specific TKIs. These observations suggest that continued investigation of rare TKI-resistant EGFR variants is warranted to identify optimal treatments for cancer.