Constitutive Signaling Activity Research Articles

A phase 1b/2 study of VS-6766 in combination cetuximab in patients (pts) with advanced KRAS mt colorectal cancer (CRC).

TPS219 Background: KRAS mutations (mts) are present in about 45% of CRC and predict lack of response to anti-EGFR therapy like cetuximab. Limited therapy options exist for pts after prior 5-FU based regimens. Regorafenib or TAS-102 is commonly used however, the modest clinical benefit, and toxicity limit their use. Novel therapies are needed for pts at this point in their disease course. KRAS G12D and G12V mts occur in 11-12% and 9-10% of CRC, respectively, whereas G12C mts occur in 3-4% of CRCs . KRAS G12C mts occur in 3-4% of CRCs. Recently, results of phase 1/2 KRYSTAL-1 study were reported. Adagrasib (a KRAS G12C inhibitor) was used with/without cetuximab in heavily pretreated CRC pts harboring KRAS G12C mts. The objective response rate (ORR) and disease control rate (DCR) was 43% and 100% (resp.) in pts receiving cetuximab and adagrasib (28 evaluable pts), and 22% and 87%, resp., in those receiving adagrasib alone (42 evaluable pts). Phase 1b CodeBreaK101 study evaluating sotorasib ( KRAS G12C inhibitor) and panitumumab (anti-EGFR) combination in KRAS G12C mt CRC showed 15.4% confirmed ORR and 26.9% unconfirmed ORR. These data are encouraging, suggesting EGFR inhibition in combination with downstream KRAS inhibition may represent important therapeutic strategy for this disease. KRAS mts lead to constitutive activation of the MAPK pathway signaling and cell activation. VS-6766 is a novel dual RAF/MEK inhibitor which has shown activity in KRAS mutated tumors. Combination of EGFR inhibition and VS-6766 may overcome resistance of KRAS mt CRC cancers to EGFR inhibition alone. Preclinically, VS-6766 and EGFR inhibition showed synergy in KRAS mt CRC cell lines, including cell lines harboring KRAS G12D and G12V mts, and CRC PDX of KRAS G12V mt CRC showed tumor regression with this combination. These data support the development of VS-6766 with anti-EGFR therapy in KRAS mt CRC warranting this phase 1 study to evaluate safety and efficacy of this combination in clinical settings. Methods: This is an open label, single arm study evaluating VS-6766 with cetuximab in pts with KRAS mt advanced CRC. Phase 1b primary endpoints include safety and tolerability, and maximum tolerated dose and recommended phase 2 dose determination. ORR is the primary endpoint of the Phase 2 study. Secondary endpoints include OS and PFS. There will be upto 4 dose levels tested. Three de-escalation doses to find the optimal cetuximab dose, and one dose escalation of VS-6766, are planned. Eligible pts include those with metastatic CRC and progression after 5-FU, oxaliplatin, irinotecan and VEGFi therapy. Based on prior studies, dermatologic, gastrointestinal, ocular and CPK elevation have been the main toxicities noted with VS-6766. The study is funded by research grants from Verastem Oncology. Cetuximab will be supplied by Eli Lilly.

Development of a degrader against oncogenic fusion protein FGFR3-TACC3

Fibroblast growth factor receptor 3-transforming acidic coiled-coil containing protein 3 (FGFR3-TACC3), which has been identified in many cancers such as glioblastoma and bladder cancer, is a potent oncogenic fusion protein that induces constitutive activation of FGFR signaling, resulting in uncontrolled cell proliferation. Although several tyrosine kinase inhibitors against FGFR are currently under development, resistance to such types of inhibitors in patients has become a concern. In this study, a chimeric molecule SNIPER(TACC3)-11 (5a) was developed and found to reduce FGFR3-TACC3 levels effectively. Compound 5a conjugated KHS108 (a TACC3 ligand) to an LCL161 derivative (11) (an inhibitor of apoptosis protein [IAP] ligand) with a PEG linker (n = 2). Mechanistical analysis showed that cellular IAP1 was required for the reduction of FGFR3-TACC3 levels. Consistent with the decrease in FGFR3-TACC3 levels, compound 5a suppressed the growth of FGFR3-TACC3 positive cells. Thus, compound 5a is a candidate therapeutic with a novel drug modality against cancers that exhibit FGFR3-TACC3-dependent proliferation and exerts pharmacological effects distinct from FGFR3 kinase inhibitors because it lacks substructures crucial for kinase inhibition.

Lung Inflammation in STING-Associated Vasculopathy with Onset in Infancy (SAVI).

STING-associated vasculopathy with onset in infancy (SAVI) is a type I interferonopathy caused by gain-of-function mutations in STING1 encoding stimulator of interferon genes (STING) protein. SAVI is characterized by severe inflammatory lung disease, a feature not observed in previously described type I interferonopathies i.e., Mendelian autoinflammatory disorders defined by constitutive activation of the type I interferon (IFN) pathway. Molecular defects in nucleic acid metabolism or sensing are central to the pathophysiology of these diseases, with such defects occurring at any step of the tightly regulated pathway of type I IFN production and signaling (e.g., exonuclease loss of function, RNA-DNA hybrid accumulation, constitutive activation of adaptor proteins such as STING). Among over 30 genotypes, SAVI and COPA syndrome, whose pathophysiology was recently linked to a constitutive activation of STING signaling, are the only type I interferonopathies presenting with predominant lung involvement. Lung disease is the leading cause of morbidity and mortality in these two disorders which do not respond to conventional immunosuppressive therapies and only partially to JAK1/2 inhibitors. In human silicosis, STING-dependent sensing of self-DNA following cell death triggered by silica exposure has been found to drive lung inflammation in mice and human models. These recent findings support a key role for STING and nucleic acid sensing in the homeostasis of intrinsic pulmonary inflammation. However, mechanisms by which monogenic defects in the STING pathway lead to pulmonary damages are not yet fully elucidated, and an improved understanding of such mechanisms is fundamental to improved future patient management. Here, we review the recent insights into the pathophysiology of SAVI and outline our current understanding of self-nucleic acid-mediated lung inflammation in humans.

Ceramides regulate defense response by binding to RbohD in Arabidopsis.

Sphingolipids, a class of bioactive lipids, play a critical role in signal transduction. Ceramides, which are central components of sphingolipid metabolism, are involved in plant development and defense. However, the mechanistic link between ceramides and downstream signaling remains unclear. Here, the mutation of alkaline ceramidase in a ceramide kinase mutant acd5 resulted in spontaneous programmed cell death early in development and was accompanied by ceramide accumulation, while other types of sphingolipids, such as long chain base, glucosylceramide, and glycosyl inositol phosphorylceramide, remained at the same level as the wild-type plants. Analysis of the transcriptome indicated that genes related to the salicylic acid (SA) pathway and oxidative stress pathway were induced dramatically in acer acd5 plants. Comparison of the level of reactive oxygen species (ROS), SA, and ceramides in the wild-type and acer acd5 plants at different developmental stages indicated that the acer acd5 mutant exhibited constitutive activation of SA and ROS signaling, which occurred simultaneously with the alteration of ceramides. Overexpressing NahG in the acer acd5 mutant could completely suppress its cell death and ceramide accumulation, while benzo-(1,2,3)-thiadiazole-7-carbothioc acid S-methyl ester treatment restored its phenotype again. Moreover, we found that the plasma membrane of acer acd5 mutant was the main site of ROS production. Ceramides accumulated in the plasma membrane of acer acd5, directly binding and activating the NADPH oxidase RbohD and promoting hydrogen peroxide generation and SA- or defense-related gene activation. Our data illustrated that ceramides play an essential role in plant defense.

Establishing a Novel Pipeline That Combines in-Silico Prediction with in-Vitro and Ex-Vivo Validation to Discover Secondary Drug Combinations Against Relapsed and/or Refractory Multiple Myeloma

Multiple myeloma (MM) is the second-most common hematological malignancy in the US. MM is an incurable, age-dependent plasma cell neoplasm with a 5-year survival rate of less than 50%. Extensive inter-individual variation in response to standard-of-care drugs like proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), drug resistance, and dose-limiting toxicities are critical problems for the treatment of MM. Clinical success in anti-myeloma treatment, therefore, warrants continuous development of novel combination therapy strategies with the explicit goal to improve the therapeutic efficacy by concomitantly targeting multiple signaling pathways.Previously, we have reported the development of an in-house computational pipeline called secDrug that applies greedy algorithm-based set-covering computational optimization method followed by a regularization technique to predict secondary drugs that can be repurposed as novel synergistic partners of standard-of-care drugs for the management of refractory/ resistant MM. Top among these secondary drugs (secDrugs) were the HSP90 inhibitor 17-AAG.In this study, we used 17-AAG as a proof of principle to establish a pipeline that integrates our in silico predictions with in vitro and ex vivo validation as well as multi-omics technologies to identify, validate, and characterize therapeutic agents that could be used either alone or in combination with standard-of-care drugs for the treatment of R/R MM patients (Figure 1).To screen and validate our in silico prediction results, we performed in vitro cytotoxicity assays using 17-AAG on a panel of human myeloma cell lines (HMCLs; in vitro model systems) that captures a wide range of biological and genetic heterogeneity representing the complexities encountered in clinical settings. These cell lines include HMCLs representing innate sensitive/resistance, >10 pairs of parental and clonally-derived PI- and IMiD-resistant pairs (P vs VR or LenR; representing acquired/emerging resistance/relapse), NRAS mutants which leads to the constitutive activation of oncogenic Ras signaling, and CRISPR-edited HSP90 knockdown cell line. Our results showed that 17-AAG has high synergistic activity in combination with PI in inducing apoptosis even in innate and acquired PI-resistant HMCLs and significantly reduces the effective dose of PI required to achieve IC 50 (Chou-Talalay's Dose Reduction Index or DRI 7±1.4). Moreover, 17-AAG+IMiD showed synergistic cell killing activity in clonally-derived IMiD resistant HMCL. Further, 17-AAG induced cell death was comparable with Hsp90 knockdown as evident from the cytotoxicity assay using PI and 17-AAG in combination in RPMI8226-wild type and RPMI-HSP90AA1 knocked down cell line. Notably, 17-AAG was strikingly effective against the NRAS-mutant cell line indicating an additional niche (NRas mutant myeloma) where 17-AAG could be most effective.Next, we performed RNA sequencing to elucidate the molecular mechanisms behind 17-AAG drug action, drug synergy, 17-AAG-induced cell death. Our gene expression profiling (GEP) followed by Ingenuity Pathway Analysis (IPA) analysis revealed protein ubiquitination, aryl hydrocarbon receptor signalling pathway as the top canonical pathways. 17-AAG induced apoptosis via mitochondrial mediated pathway in myeloma. 17-AAG exerts its cytotoxic effect by activating intrinsic pathway of apoptosis which we further confirmed through the increase in reactive oxygen species generation and decrease in mitochondrial membrane potential. 17-AAG was also effective in reducing the expression of hallmarks of MM such as p65/NF-kB, IRF4, c-Myc. Finally, we performed mass cytometry (CyTOF; Cytometry by time of flight) on primary bone-marrow cells (PMCs) from myeloma patients for further validation of proteomic signatures at the single-cell level. CyTOF analysis confirmed 17-AAG-induced cell death and key changes in MM-specific proteomic markers. 17-AAG treated PMCs showed elevated cleaved caspase levels and down-regulation of IRF4 and phospho-STAT3. GEP and CyTOF results were confirmed using immunoblotting assays.Together, our study demonstrates a unique pipeline for drug repositioning that has the potential to revolutionize clinical decision-making by minimizing the number of drugs required for discovering successful combination chemotherapy regimens against drug-resistant myeloma. [Display omitted] DisclosuresKumar: BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Beigene: Consultancy; Oncopeptides: Consultancy; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche-Genentech: Consultancy, Research Funding; Bluebird Bio: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding.

MTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

TRPC3, an underestimated, universal pacemaker channel?

Transient receptor potential channel canonical 3 (TRPC3) is a cation channel with poor Ca2+ selectivity and significant constitutive activity. One of the channels’ features is its striking ability to couple in a surprisingly versatile manner to different down-stream signaling pathways, thereby serving cellular functions in a tissue specific manner. Expression of this protein is prominent in excitable cells, and its activity has repeatedly been implicated in electrical pacemaking. Previous studies demonstrated a linkage between constitutive activity of TRPC3 and neuronal firing in hippocampus and cerebellum. A most recent report from the Park laboratory corroborates the concept of TRPC3 functioning as a critical element in the neuronal pacemaking machinery for dopaminergic neurons of substantia nigra. Conclusively, mechanistic coupling between TRPC3 activity and firing frequency appears evident for different types of neurons, highlighting the potential of TRPC3 as a universal as well as multifunctional pacemaker channel.

The HIF1\u03b1-PDGFD-PDGFR\u03b1 axis controls glioblastoma growth at normoxia/mild-hypoxia and confers sensitivity to targeted therapy by echinomycin

BackgroundGlioblastoma multiforme (GBM), a lethal brain tumor, remains the most daunting challenge in cancer therapy. Overexpression and constitutive activation of PDGFs and PDGFRα are observed in most GBM; however, available inhibitors targeting isolated signaling pathways are minimally effective. Therefore, better understanding of crucial mechanisms underlying GBM is needed for developing more effective targeted therapies.MethodsTarget genes controlled by HIF1α in GBM were identified by analysis of TCGA database and by RNA-sequencing of GBM cells with HIF1α knockout by sgRNA-Cas9 method. Functional roles of HIF1α, PDGFs and PDGFRs were elucidated by loss- or gain-of-function assays or chemical inhibitors, and compared in response to oxygen tension. Pharmacological efficacy and gene expression in mice with intracranial xenografts of primary GBM were analyzed by bioluminescence imaging and immunofluorescence.ResultsHIF1α binds the PDGFD proximal promoter and PDGFRA intron enhancers in GBM cells under normoxia or mild-hypoxia to induce their expression and maintain constitutive activation of AKT signaling, which in turn increases HIF1α protein level and activity. Paradoxically, severe hypoxia abrogates PDGFRα expression despite enhancing HIF1α accumulation and corresponding PDGF-D expression. Knockout of HIF1A, PDGFD or PDGFRA in U251 cells inhibits cell growth and invasion in vitro and eradicates tumor growth in vivo. HIF1A knockdown in primary GBM extends survival of xenograft mice, whereas PDGFD overexpression in GL261 shortens survival. HIF1α inhibitor Echinomycin induces GBM cell apoptosis and effectively inhibits growth of GBM in vivo by simultaneously targeting HIF1α-PDGFD/PDGFRα-AKT feedforward pathway.ConclusionsHIF1α orchestrates expression of PDGF-D and PDGFRα for constitutive activation of AKT pathway and is crucial for GBM malignancy. Therefore, therapies targeting HIF1α should provide an effective treatment for GBM.

MTOR-activating mutations of RRAGD cause a novel syndrome combining kidney tubulopathy and cardiomyopathy

Advances in genetic techniques have resulted in the identification of rare inherited disorders of renal magnesium and salt handling. Nevertheless, ∼20% of all patients with primary kidney tubulopathy have no diagnosis. We explore a large multicentric cohort presenting with a novel inherited salt-losing tubulopathy mostly characterized by hypomagnesemia, combined with early-onset dilated cardiomyopathy (DCM). Whole exome and genome sequencings were performed with various subsequent functional analyses of identified RRAGD variants in vitro . In 8 children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt-wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo . Six of these patients additionally suffered from DCM, and a heart transplantation was performed in 3 of them before the age of 25y. A dominant variant in RRAGD was simultaneously identified in 8 members of a large family, which segregated with a similar renal magnesium-losing phenotype. No DCM was detected in this family. RRAGD encodes Rag GTPase D mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron includes the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro . Our findings establish a novel diseased phenotype combining kidney tubulopathy and cardiomyopathy caused by an activation of mTOR signaling suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

Fibrous Caps in Atherosclerosis Form by Notch-Dependent Mechanisms Common to Arterial Media Development.

Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Structural basis for the constitutive activity and immunomodulatory properties of the Epstein-Barr virus-encoded G protein-coupled receptor BILF1

Epstein-Barr virus (EBV) encodes a G protein-coupled receptor (GPCR) termed BILF1 that is essential for EBV-mediated immunosuppression and oncogenesis. BILF1 couples with inhibitory G protein (Gi), the major intracellular signaling effector for human chemokine receptors, and exhibits constitutive signaling activity; the ligand(s) for BILF1 are unknown. We studied the origins of BILF1's constitutive activity through structure determination of BILF1 bound to the inhibitory G protein (Gi) heterotrimer. The 3.2-Å resolution cryo-electron microscopy structure revealed an extracellular loop within BILF1 that blocked the typical chemokine binding site, suggesting ligand-autonomous receptor activation. Rather, amino acid substitutions within BILF1 transmembrane regions at hallmark ligand-activated class A GPCR "microswitches" stabilized a constitutively active BILF1 conformation for Gi coupling in a ligand-independent fashion. Thus, the constitutive activity of BILF1 promotes immunosuppression and virulence independent of ligand availability, with implications for the function of GPCRs encoded by related viruses and for therapeutic targeting of EBV.

Enhanced Efficacy of the SYK Inhibitor Entospletinib and Chemotherapy in Infant Acute Lymphoblastic Leukemia

Background: Chemotherapy resistance and subsequent relapse remain a major cause of childhood cancer mortality, particularly for infants (<12 months of age) with KMT2A -rearranged (R) B-cell acute lymphoblastic leukemia (B-ALL), who have extremely poor event-free survival (EFS). Addition of kinase inhibitors to chemotherapy has markedly improved outcomes for patients with some genetic subsets of B-ALL, such as BCR-ABL1 -R (Ph+) ALL. Unfortunately, addition of the FMS-like tyrosine kinase 3 (FLT3) inhibitor lestaurtinib did not improve 3-year EFS for infants with KMT2A -R and FLT3-overexpressing B-ALL in the Children's Oncology Group trial AALL0631 (Brown et al, SIOP 2016), indicating continued need for alternative therapeutic strategies. We recently observed constitutive phosphorylation (p) of spleen tyrosine kinase (SYK) at tyrosine 323 in infant ALL specimens (Figure 1A) and hypothesized that activated signaling is targetable with the selective SYK inhibitor entospletinib (ENTO). We further hypothesized that combining ENTO with chemotherapy would have superior activity in comparison to single-agent treatments.Methods: To test these hypotheses, we established patient-derived xenograft (PDX) models of KMT2A -R (n=7) and non- KMT2A -R (n=4) infant ALL in NSG mice using diagnostic and relapse bone marrow specimens from patients treated on or as per the AALL0631 trial protocol. Total and phosphorylated (p) SYK and other phosphoprotein levels were assessed by Simple WesternTM analysis of splenic lysates from ALL-engrafted PDX mice to identify leukemias with constitutive SYK signaling activation. Infant ALL PDX models were then treated with control mouse chow or chow containing 0.07% ENTO ad libitum, 0.25 mg/kg vincristine (VCR) dosed intraperitoneally once weekly, or a combination of ENTO and VCR for up to 28 days to determine treatment efficacy. Cohorts of additional infant ALL PDX mice were treated for 72 hours to assess plasma levels and pharmacodynamic effects of ENTO administration.Results: Constitutive SYK signaling was observed in several models of de novo KMT2A -R and non- KMT2A -R B-ALL. ENTO treatment of KMT2A -R ALL PDX models ALL3103 with t(9;11) and ALL135 with t(11;19) inhibited leukemic cell burden in vivo versus control animals by flow cytometric and immunohistochemical (IHC) analyses of murine spleens (representative data in Figure 1B). Importantly, combined ENTO and VCR treatment resulted in a greater reduction in ALL cell number, which was confirmed by human CD19+ IHC analysis of murine spleens and bone marrow. ENTO concentrations were maintained throughout study duration with terminal free fraction plasma levels of 61 and 230 nM (ALL3103) and 89 and 214 nM (ALL135) for ENTO and ENTO/VCR treatment arms, respectively. Evaluation of SYK pathway modulation in vivo demonstrated inhibition of pSYKY323 in human ALL cells within murine spleens that correlated with the level of ENTO exposure of treated mice (ALL3103 PDX; Spearman correlation of -0.63, p<0.05). No alterations in total SYK protein were observed. RNA sequencing of ALL3103 PDX tissues showed that ENTO treatment was associated with downregulation of MYC and of cell cycle and DNA damage gene expression signatures.Conclusions: Constitutive activation of SYK signaling occurs in infant KMT2A -R B-ALL. Entospletinib treatment of infant B-ALL PDX models potently inhibited the SYK pathway and decreased leukemic cell burden in vivo . Superior anti-ALL activity was observed with combined ENTO and VCR compared to ENTO alone. Additional studies are ongoing to determine the activity of ENTO in non- KMT2A -R and other ALL PDX models. [Display omitted] DisclosuresYahiaoui:Gilead Sciences, Inc.: Employment, Equity Ownership. Lopez:Gilead Sciences, Inc.: Employment, Equity Ownership. Mikaelian:Gilead Sciences, Inc.: Employment, Equity Ownership. Tannheimer:Gilead Sciences, Inc.: Employment, Equity Ownership. Forslund:Gilead Sciences, Inc.: Employment, Equity Ownership. Tasian:Gilead Sciences, Inc.: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Combination of IRAK4 Inhibitor CA-4948 with BCL2 Inhibitor Venetoclax Induces Tumor Regression in an ABC-DLBCL Xenograft Model

IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of diffuse large B-cell lymphoma (DLBCL) cases. The MYD88- L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. In Waldenstrom macroglobulinemia (WM), the MYD88- L265P activating mutation is present in >90% of cases. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL and WM.We are developing a novel IRAK4 inhibitor, CA-4948, as a therapeutic agent for non-Hodgkin lymphoma (NHL) and hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 is a reversible kinase inhibitor that modulates IRAK4 function in both the toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling cascades, and demonstrates pharmacodynamic and antitumor activity in in vitro and in vivo nonclinical models. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88 -L265P xenograft tumor models using cell lines OCI-LY3 and OCI-Ly10 and patient-derived tumors.Activating mutations in the B-cell receptor (BCR) signaling pathway are frequently present in various NHL subtypes, leading to activation of NF-kB signaling, and growth and survival pathways. Dysregulation of both TLR and BCR pathways suggest that targeting both pathways will be required for improved therapeutic responses in NHL. Here, we report efficacy results from combined treatment of CA-4948 and the BCL2 inhibitor venetoclax in an ABC-DLBCL xenograft tumor model.We performed an in vivo study in female SCID Beige mice implanted with the ABC-DLBCL cell line OCI-Ly10, which harbors TLR (MYD88 -L265P) and BCR (CD79A ITAM) pathway activating mutations. OCI-Ly10 tumor-bearing mice were treated with CA-4948 (50 mg/kg, qd, po), venetoclax (75 mg/kg, qd, po) or the combination for 21 continuous days. Single agent treatment of CA-4948 and venetoclax exhibited moderate tumor growth inhibition (TGI) of 63% and 71%, respectively, and the combination demonstrated tumor regression. The combination was tolerated at the end of treatment without body weight loss relative to predose weight. After the 21-day drug treatment period, the mice were given a 19-day dosing holiday. Within 5 days of the holiday rapid tumor growth occurred in the previously treated single drug-treated mice and this rapid growth continued for the remainder of the dosing holiday. In contrast, tumors from the combination drug-treated mice did not commence detectable regrowth until the 10th day of the drug holiday and proceeded with a slow growth rate for the subsequent 9 days.After the 19-day drug holiday, dosing of the respective drug treatments was reinstated to each cohort. After 7 days of re-dosing, the tumors in each cohort exhibited antitumor responses to respective drug treatments with the CA-4948 plus venetoclax combination again driving the tumors into regression. In summary, CA-4948 plus venetoclax was a well-tolerated drug combination that exhibited a much greater in vivo antitumor response as compared to the single agent treatments. These results underscore the therapeutic potential of targeted IRAK4 kinase inhibition by CA-4948 in combination with other targeted agents for the treatment of NHL. DisclosuresBooher:Curis, Inc: Employment, Equity Ownership. Dellarocca:Curis, Inc: Employment, Equity Ownership. Atoyan:Curis, Inc: Employment, Equity Ownership. Borek:Curis, Inc: Employment, Equity Ownership. Samson:Curis, Inc: Employment, Equity Ownership. Tuck:Curis, Inc: Employment, Equity Ownership.

PI3Kinase Inactivation through Combined Loss of Catalytic Subunits p110α, p110β and p110δ Derails Hematopoietic Homeostasis

PI3Kinase is a lipid kinase that is involved in diverse cellular processes including cell growth, survival, differentiation and cell cycle. Deregulation of PI3K pathway has been associated with wide variety of cancers including several hematological disorders such as acute myeloid leukemia (AML). Hematopoietic growth factors such as Fms Related Tyrosine Kinase 3 (FLT3) ligand, stem cell factor (SCF), erythropoietin (EPO), and thrombopoietin (TPO) activate PI3K signaling pathway via tyrosine kinase receptors, growth factor receptors and G-protein coupled receptors. In mouse models of abrogation of PI3K signaling, involving AKT deletion, impairs the self-renewal potential of hematopoietic stem cells (HSCs). In contrast, mice models with constitutive activation of PI3K signaling, involving deletion of PTEN phosphatase, causes increased cycling of HSCs. PI3K regulatory subunits, P85α and P85β, are critical for fetal liver hematopoiesis. However, the role of PI3K in adult hematopoiesis and contribution of catalytic subunits of PI3K to hematopoietic homeostasis is not well studied. Given that HSCs express all three catalytic subunits of PI3K, we developed a mouse model for ablated PI3K pathway by depleting PI3K catalytic subunits p110α, p110β and p110δ. p110α and p110β subunits were conditionally deleted via MX-Cre system upon Poly-IC injection, and p110δ inactivation was achieved through p110δ inactivating knock-in mutation. Mouse tissues were harvested upon 5 weeks post poly-IC, at which time point the mice appeared moribund and were therefore analyzed for various hematopoietic lineages. These mice were anemic and peripheral blood analysis showed leukopenia, neutropenia and anemia with significantly reduced WBC, neutrophils, RBC, hemoglobin, hematocrits and platelet counts. Mice depleted of p110α, β and δ subunits-p110 triple knock out mice (p110-TKO) - demonstrated significantly reduced spleen size, reduced bone marrow cellularity with anemic bones relative to wild type mice. Hematopoietic stem cell compartment in p110-TKO mice showed increased frequency in Lineage-veSca1+veKit+ve (LSK) population as well as an increase in more primitive HSCs defined as SLAM cells (CD150+veCD48-ve). In contrast, there was reduction in total number of common lymphoid progenitors (CLP), common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP) in the bone marrow. These mice also showed dramatically reduced total B-cells and significantly reduced T-cells. However, these mice show increased frequency of GR1-Mac1 positive granulocytes in all hematopoietic compartments. Erythroid compartment was analyzed for various stages of red blood cell development using Ter119 and CD71 markers. Hampered RBC development was observed in p110-TKO mice with significantly increased immature erythroid cells (Ter119lowCD71low) in the bone marrow, spleen and peripheral blood compartments, with significantly reduced terminally differentiated cells (Ter119+ve only). HSC self-renewal studies involving competitive repopulation assay showed that recipient mice have significantly reduced p110-TKO donor chimerism. Earlier studies from others have reported that p110α and p110δ subunits are dispensable for adult HSC function, however p110α is required for terminal erythroblast maturation and p110δ is required for leukocyte development. Our studies revealed that lack of all three catalytic subunits of PI3Kinase significantly impairs hematopoietic homeostasis with hampered stem cell development, erythroid cell development and a complete shut down in B-cell production. DisclosuresNo relevant conflicts of interest to declare.

Targeted Therapy with BET Protein and β Catenin Antagonists Against Ruxolitinib Resistant Post-Myeloproliferative Neoplasm (MPN) Secondary (s) AML Cells

Hematopoietic stem/progenitor cells (HPCs) in myeloproliferative neoplasms with myelofibrosis (MPN-MF) exhibit mutations in the JAK2, c-MPL, or calreticulin (CALR) gene and display constitutive activation of JAK-STAT signaling. In MPN-MF, secondary AML (sAML) occurs in up to 20% of patients. Ruxolitinib (Rux), a type I, ATP-competitive, dual JAK1/2 inhibitor (JAKi), confers notable clinical benefit in MPN-MF, but exhibits only modest activity and does not significantly impact the clinical outcome in post-MPN sAML. Because standard chemotherapy is also relatively ineffective, there is an urgent need to develop and test novel agents for the treatment of sAML. We previously reported that, although they lack additional JAK2 mutations compared to their sensitive parental counterparts, in vitro generated Rux-resistant, JAKi-persister/resistant HEL-92.1.7 and SET2 cells (JAKi-P/R) cells exhibit 3 to 10-fold lower sensitivity to Rux, and display reactivation of JAK-STAT signaling due to trans-phosphorylation of JAK2 by JAK1 or TYK2 tyrosine kinases (TKs). We hypothesized that the relative resistance to JAKi in sAML cells would be governed by a dysregulated transcriptome and dependent on the activity of the chromatin reader BET proteins (BETPs), including BRD4. Therefore, we determined the lethal activity of BETP PROTACs (proteolysis targeting chimeras) ARV-825, and its pharmacologically superior version ARV-771, that proteasomally degrade BETPs, against JAKi-P/R sAML cells. ARV-771 recruits the E3 ligase VHL (Von Hippel-Lindau) to polyubiquitylate and mediate multiple rounds of sub-stoichiometric catalysis, leading to efficient and prolonged degradation and depletion of BETPs, including BRD4 (> 90%) in the sAML cells. Treatment with BETP-PROTAC (50 to 250 nM) markedly depleted BRD4 and BRD2, as well as caused robust attenuation of the levels of MYC, p-STAT5, Bcl-xL, PIM1 and CDK6 in JAKi-P/R sAML cells (Leukemia, 2017, PMID: 28042144). Concomitantly, BETP-PROTAC treatment mediated profound growth inhibition and apoptosis of Rux-sensitive and JAKi-P/R cultured and patient-derived CD34+ sAML cells. This translated into a significantly reduced sAML burden and improved survival, without toxicity, in the luciferase-transduced immune depleted (NSG) mice treated with ARV-771 (30 mg/kg/day for 3 weeks) versus vehicle control. In myeloid malignancies, canonical WNT-β catenin signaling is essential for survival, growth and self-renewal of leukemia stem and blast progenitor cells (BPCs). Notably, we discovered that, as compared to their sensitive controls, JAKi-P/R sAML cells exhibited higher levels of p-AKT, p-GSK3-β and increased nuclear localization of β-catenin (by confocal microscopy), associated with increased levels of β-catenin-TCF4 targets, MYC and survivin. In a previous report, we had demonstrated that treatment with the β-catenin antagonist BC-2059 that degrades and depletes nuclear levels of β-catenin and inhibits β-catenin/TCF4 transcriptional targets, resulted in apoptosis of AML stem progenitor cells (Leukemia, 2015;29:1267-78). Consistent with this, we determined that BC-2059 (50 to 100 nM) also markedly attenuated nuclear β-catenin levels, reduced expression of the β-catenin/TCF4 targets MYC and survivin, and induced in vitro apoptosis of JAKi-P/R sAML and PD CD34+ sAML cells. Co-treatment with BC-2059 and Rux synergistically induced in vitro apoptosis (combination indices < 1.0) of sAML cells, and combined therapy for 3 weeks with BC-2059 (30 mg/kg/day) and Rux (30 mg/kg/day) significantly reduced sAML burden and improved survival of NSG mice engrafted with sAML cells (p < 0.01). Recently, resistance to BETP inhibitors that evict BETPs from chromatin was shown to be at least in part due to increased activity of WNT-β-catenin/TCF4 and the increased c-Myc expression. Based on this, we also determined the in vitro and in vivo effects of co-treatment with BETP-PROTAC and BC-2059 against sAML cells. Compared to each drug alone, co-treatment with BETP-PROTAC exerted synergistic in vitro lethality against JAKi sensitive and JAKi-P/R sAML cells. These findings strongly support further in vivo development and testing of BETP-PROTAC and β-catenin antagonist-based combinations against post-MPN sAML. DisclosuresQian:4Arvinas, LLC. New Haven, CT: Employment. Raina:4Arvinas, LLC. New Haven, CT: Employment. Verstovsek:Celgene: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Lilly Oncology: Research Funding; Pfizer: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding; Genentech: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Incyte: Research Funding; Lilly Oncology: Research Funding; Promedior: Research Funding; Roche: Research Funding; Roche: Research Funding; Astrazeneca: Research Funding; Blueprint Medicines Corp: Research Funding; Genentech: Research Funding; Blueprint Medicines Corp: Research Funding; Bristol Myers Squibb: Research Funding; NS Pharma: Research Funding.

A Novel JAK2 Mutation Confers Cytokine-Independence in the Absence of a Type I Dimeric Cytokine Receptor

JAK2 fusions reported in hematologic malignancies are often associated with eosinophilia. In addition to JAK2 fusions such as PCM1-JAK2, BCR-JAK2 and ETV6-JAK2, the JAK2V617Fpoint mutant has been recurrently observed in patients with hypereosinophilic syndrome (HES), occurring in ~4% of patients with hypereosinophilia of unknown significance (Schwaab, Am J Hematol, 2015).Here, we describe a novel JAK2 mutation in a 69 year-old woman with a history of immune thrombocytopenia referred for steroid-refractory HES. Peripheral blood eosinophilia was present for at least five years, with absolute eosinophil counts ranging up to 17,500/μL. Bone marrow biopsy revealed hypercellular marrow with trilineage hematopoiesis, increased atypical megakaryocytes, and markedly increased eosinophils with abnormal morphology, consistent with myeloproliferative neoplasm. Results from an eosinophilia FISH panel for FIP1L1/CHIC2/PDGFRα, FGFR, PDGFRβ, and CBFβ fusions were negative, as were T-cell clonality studies by next-generation sequencing (NGS). An NGS-based myeloid malignancies panel revealed a novel mutation in JAK2 consisting of a ten base deletion and insertion of a single thymine (JAK2 c.1747_1756delinsT, p.Leu583_Ala586delinsSer;JAK2indel). Treatment with ruxolitinib improved the peripheral blood eosinophilia, but dose intensity was limited by thrombocytopenia (Table 1).For transformation of cells to cytokine-independence, JAK2V617F requires co-expression of a type I dimeric cytokine receptor, such as Epo-R, to enable downstream signaling in the absence of growth factors (Lu et al, Proc Natl Acad Sci, 2005). To characterize the signaling properties of the new JAK2indel mutant, we cloned JAK2WT, JAK2V617F, and JAK2indel into the MSCV-IRES-GFP (MIG) retroviral vector. IL-3-dependent Ba/F3 cells lacking type I cytokine receptors were infected with retrovirus to assess cytokine independence.Parental Ba/F3 cells and those transduced with JAK2WT, JAK2V617F, or JAK2indel proliferated at similar rates when grown in WEHI conditioned medium (CM). However, upon IL-3 (WEHI) withdrawal, only GFP-positive JAK2indel Ba/F3 cells demonstrated growth factor independence (n=3). Furthermore, parental and GFP-sorted JAK2WT- and JAK2V617F-expressingBa/F3 cells display acceleration of growth when cultured in graded dilutions of WEHI CM, while growth of JAK2indel Ba/F3 cells is not further enhanced by WEHI CM. JAK2indel-expressing Ba/F3 cells also demonstrated cytokine-independent growth in colony assays, whereas parental, JAK2WT-, and JAK2V617F-expressing cells displayed little to no colony formation in the absence of cytokines. Immunoblot analyses revealed that after 4 hours of culture ± IL-3, all cells grown in IL-3 demonstrated activation of STAT5 and ERK, but only JAK2indel Ba/F3 cells demonstrated cytokine-independent signaling activation.JAK2indel activates STAT5 and ERK without co-expression of a type I cytokine receptor, implicating a mechanism of JAK2 kinase activation that is distinct from JAK2V617F. Unexpectedly, preliminary structural analysis suggests that the conformational change induced by JAK2indel resembles that of JAK2V617F. Mutation of glutamic acid 596 to arginine (E596R) has been shown to abolish the constitutive activity of JAK2V617F via modulation of the C-terminal residues of the SH2-JH2 linker, thereby restoring pseudokinase domain autoinhibitory function (Leroy et al, Biochem J, 2016). To test whether this regulatory mechanism is preserved in JAK2indel, we introduced E596R into JAK2indel and are currently characterizing JAK2indel/E596R in cellular and biochemical assays. Additional modeling and biochemical work is underway to clarify whether JAK2indel confers a new functionality that supports signaling activation in the absence of a type I cytokine receptor.In summary we report a novel JAK2 mutation in a patient with myeloproliferative-variant HES that results in cytokine-independent growth, is associated with STAT5 and ERK phosphorylation and may be employing an alternative mechanism to achieve constitutive activation of signaling. The fact that the patient's eosinophilia improved with ruxolitinib treatment illustrates the importance of performing NGS in patients with HES to identify potentially targetable genetic lesions. [Display omitted] DisclosuresDeininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; ARIAD: Consultancy.

Targeting the HIF-1α-IGFBP2 axis therapeutically reduces IGF1-AKT signaling and blocks the growth and metastasis of relapsed anaplastic Wilms tumor.

For patients with anaplastic Wilms tumor (WiT), metastasis and recurrence are common, and prognosis is generally poor. Novel therapies are needed to improve outcomes for patients with this high-risk WiT. A potential contributor to WiT development is constitutive activation of AKT by insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) signaling pathway, but the complete underlying mechanism remains unclear. Here, we demonstrate that the hypoxia-inducible factor 1α (HIF-1α)-IGF binding protein 2 (IGFBP2) axis and the tumor-specific IGF1A are key players for constitutive activation of IGF1-AKT signaling leading to the tumor malignancy. HIF-1α and IGFBP2 are highly expressed in a majority of WiT patient samples. Deficiency of either HIF-1α or IGFBP2 or IGF1 in the tumor cells significantly impairs tumor growth and nearly abrogates metastasis in xenografted mice. Pharmacologic targeting of HIF-1α by echinomycin delivered via nanoliposomes can efficiently restrain growth and metastasis of patient-derived relapsed anaplastic WiT xenografts. Liposomal echinomycin is more potent and effective in inhibiting WiT growth than vincristine in an anaplastic WiT mouse model, and eliminates metastasis by suppressing HIF-1α targets and the HIF-1α-IGFBP2 axis, which governs IGF1-AKT signaling.

Rigosertib inhibits MEK1-ERK pathway and alleviates lipopolysaccharide-induced sepsis.

BackgroundHere, by using the lipopolysaccharide (LPS)‐induced mice sepsis model, we treated septic wild‐type (WT) mice or MEK1DD mice with rigosertib to evaluate its prospective effects on sepsis.MethodsWe also generated macrophages derived from bone marrow from WT or MEK1DD mice. These macrophages were pretreated with rigosertib and then induced with LPS or poly I:C.ResultsRigosertib suppressed LPS or poly I:C‐induced expression of inflammatory cytokines (tumor necrosis factor‐alpha [TNF‐α] and interleukin‐6 [IL‐6], and IL‐23) in WT bone marrow–derived macrophages while failed to affect the upregulation of TNF‐α and IL‐6 in LPS‐treated bone marrow–derived macrophages from MEK1DD mice. Rigosertib promoted survival rate, ameliorated lung injury, and reduced inflammatory cytokine levels in serum of WT septic mice.ConclusionIn contrast, the effects of rigosertib on sepsis were abrogated in septic MEK1DD mice, which had inducible constitutive activation of MEK1 signaling. Rigosertib alleviated LPS‐induced sepsis inhibits MEK1/ERK signaling pathway.

Current Concepts of Pathogenesis and Treatment of Philadelphia Chromosome-Negative Myeloproliferative Neoplasms.

Philadelphia chromosome-negative myeloproliferative neoplasms are hematopoietic stem cell disorders characterized by dysregulated proliferation of mature myeloid blood cells. They can present as polycythemia vera, essential thrombocythemia, or myelofibrosis and are characterized by constitutive activation of JAK2 signaling. They share a propensity for thrombo-hemorrhagic complications and the risk of progression to acute myeloid leukemia. Attention has also been drawn to JAK2 mutant clonal hematopoiesis of indeterminate potential as a possible precursor state of MPN. Insight into the pathogenesis as well as options for the treatment of MPN has increased in the last years thanks to modern sequencing technologies and functional studies. Mutational analysis provides information on the oncogenic driver mutations in JAK2, CALR, or MPL in the majority of MPN patients. In addition, molecular markers enable more detailed prognostication and provide guidance for therapeutic decisions. While JAK2 inhibitors represent a standard of care for MF and resistant/refractory PV, allogeneic hematopoietic stem cell transplantation remains the only therapy with a curative potential in MPN so far but is reserved to a subset of patients. Thus, novel concepts for therapy are an important need, particularly in MF. Novel JAK2 inhibitors, combination therapy approaches with ruxolitinib, as well as therapeutic approaches addressing new molecular targets are in development. Current standards and recent advantages are discussed in this review.