JAK Kinase Inhibitors Research Articles

1064 JAK kinase inhibitors efficiaous in models of murine contact hypersensitivity

Murine models of dermal contact dermatitis have been used as preclinical screening tools to select compounds for topical therapy of human inflammatory diseases such as alopecia areata, vitiligo and atopic dermatitis. All of these diseases are known to be driven by JAK3/common gamma chain (gc) dependent cytokines (e.g., IL-4, IL-15), whose signaling is controlled by JAK1 and JAK3 kinases. We have synthesized soft inhibitors of JAK1/3 that are designed for high skin permeability with minimal systemic stability. Soft, topical JAK inhibitors offer the potential to treat patients, including those with mild to moderate disease, for whom systemic JAK kinase inhibitors might not be ideal. These compounds have been tested topically in a murine contact hypersensitivity model. In this model, animals are systemically immunized to the hapten 2,4-dinitrofluorobenzene (DNFB), followed one week later by topical challenge to the ear, resulting in swelling. JAK kinase inhibitors applied prior to the challenge dose of DNFB can inhibit the swelling, if applied in the appropriate vehicle. Swelling was significantly reduced by 50-70%, with P < 0.05 (unpaired T test). Promising molecules are being profiled with in vitro human/pig skin penetration models (Franz Chamber), using multiple formulations. Using these data, appropriate drug concentration and vehicle(s) will be chosen and murine findings are being extended in the minipig DNFB model as the architecture of minipig skin much more closely resembles that of human skin. Details of the histology and cytokine dependence of these models, along with the local and systemic PK of the drugs studied, will be presented. These data will inform progression to the study of human patients with dermatological autoimmune diseases.

Structure-activity studies of peptidomimetics based on kinase-inhibitory region of suppressors of cytokine signaling 1.

Suppressors of Cytokine Signaling (SOCS) proteins are negative regulators of JAK proteins that are receptor-associated tyrosine kinases, which play key roles in the phosphorylation and subsequent activation of several transcription factors named STATs. Unlike the other SOCS proteins, SOCS1 and 3 show, in the N-terminal portion, a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Drug discovery processes of compounds based on KIR sequence demonstrated promising in functional in vitro and in inflammatory animal models and we recently developed a peptidomimetic called PS5, as lead compound. Here, we investigated the cellular ability of PS5 to mimic SOCS1 biological functions in vascular smooth muscle cells and simultaneously we set up a new binding assay for the screening and identification of JAK2 binders based on a SPR experiment that revealed more robust with respect to previous ELISAs. On this basis, we designed several peptidomimetics bearing new structural constraints that were analyzed in both affinities toward JAK2 and conformational features through Circular Dichroism and NMR spectroscopies. Introduced chemical modifications provided an enhancement of serum stabilities of new sequences that could aid the design of future mimetic molecules of SOCS1 as novel anti-inflammatory compounds.

EFFECT OF TOFACITINIB ON IGE-MEDIATED DEGRANULATION OF LAD2 HUMAN MAST CELLS AND PGD2 PRODUCTION

Tofacitinib is an inhibitor of the enzymes JAK1 and JAK3, and can interfere with the JAK-STAT signaling pathway, transmission of extracellular information into the cell nucleus and DNA transcription. Tofacitinib is approved for use in rheumatoid arthritis and is being explored for use in psoriasis, colitis, atopic dermatitis and ankylosing spondylitis. Recently, tofacitinib was said to lead to symptomatic improvement in two patients diagnosed with mast cell activation syndrome. This prompted us to examine the effect of tofacitinib on the biologic reactivity of the human mast cell line, LAD2. In preliminary experiments, cultured LAD2 cells were incubated overnight with biotinylated human IgE. Cells were washed and then incubated with 1, 10, 100, 1000 nM tofacitinib for 1 hour. FcepsilonRI crosslinking was next performed with streptavidin, and degranulation monitored by the release of beta-hexosaminidase (b-Hex). LAD2 cells were also incubated with tofacitinib followed by stimulation with PMA, degranulation and release of b-Hex. The effect on PgD2 production was measured 30 minutes following IgE mediated mast cell activation. In LAD2 cells, b-Hex release following FcepsilonRI crosslinking or PMA stimulation remained consistently high (50-60%) in the presence of tofacitinib with no dose-response reduction in degranulation. Similarly, tofacitinib had no effect on PgD2 release. The JAK kinase inhibitor tofacitinib had no effect on LAD2 human mast cell degranulation or PgD2 production. Additional experiments are underway including an examination of the effects of tofacitinib on LAD2 cytokine production, adhesion and proliferation.

Application of a phenotypic drug discovery strategy to identify biological and chemical starting points for inhibition of TSLP production in lung epithelial cells.

Thymic stromal lymphopoietin (TSLP) is a cytokine released by human lung epithelium in response to external insult. Considered as a master switch in T helper 2 lymphocyte (Th2) mediated responses, TSLP is believed to play a key role in allergic diseases including asthma. The aim of this study was to use a phenotypic approach to identify new biological and chemical starting points for inhibition of TSLP production in human bronchial epithelial cells (NHBE), with the objective of reducing Th2-mediated airway inflammation. To this end, a phenotypic screen was performed using poly I:C / IL-4 stimulated NHBE cells interrogated with a 44,974 compound library. As a result, 85 hits which downregulated TSLP protein and mRNA levels were identified and a representative subset of 7 hits was selected for further characterization. These molecules inhibited the activity of several members of the MAPK, PI3K and tyrosine kinase families and some of them have been reported as modulators of cellular phenotypic endpoints like cell-cell contacts, microtubule polymerization and caspase activation. Characterization of the biological profile of the hits suggested that mTOR could be a key activity involved in the regulation of TSLP production in NHBE cells. Among other targeted kinases, inhibition of p38 MAPK and JAK kinases showed different degrees of correlation with TSLP downregulation, while Syk kinase did not seem to be related. Overall, inhibition of TSLP production by the selected hits, rather than resulting from inhibition of single isolated targets, appeared to be due to a combination of activities with different levels of relevance. Finally, a hit expansion exercise yielded additional active compounds that could be amenable to further optimization, providing an opportunity to dissociate TSLP inhibition from other non-desired activities. This study illustrates the potential of phenotypic drug discovery to complement target based approaches by providing new chemistry and biology leads.

Клинический случай IgG4-ассоциированного заболевания

IgG4-related disease is an extremely rare disorder, сcharacterized by the inflammatory infiltration of various organs, with predominance of IgG4-positive plasma cells, fibrosis and high IgG4 plasma concentration. Treatment regiments have not been optimized, especially in children. Here we discuss a case of IgG4-related disease of the orbit in a 13 year old boy and our new treatment approach involving JAK-kinase inhibitor ruxolitininb. The article also presents analysis of the state of the problem of IgG4-related disease it he world, including terminology, historical references, pathogenesis, diagnostic problems and clinical manifestations.

Redundant JAK, SRC and PI3 Kinase Signaling Pathways Regulate Cell Survival in Human Ph-like ALL Cell Lines and Primary Cells

▪Background: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk disease with a frequency of about 20% in patients of all ages. Ph-like ALL can be characterized in different groups based on their chromosomal rearrangements. The most common rearrangements involve CRLF2, ABL1, and EPOR and lead to activation of JAK kinase dependent activation of STAT5, PI3 kinase and other signaling pathways. We have previously shown that Ph-like ALL is a high-risk disease with a poor prognosis in older adults. The focus of this study is to further understand the biology of Ph-like ALL and how the cells develop drug resistance by modulating their signaling pathways to escape apoptosis.Results: We and others have identified activated STAT5 and AKT signaling in Ph-like ALL cell lines and patient samples. Interestingly, inhibition of the JAK-STAT signaling pathway using JAK-kinase inhibitors abrogated short term STA5 signaling as well as AKT signaling, but Ph-like ALL cells showed only a mild apoptotic effect or arrest in cell proliferation in vitro. Furthermore, even though xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with ruxolitinib, a JAK1/2 inhibitor, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. Based on these results we hypothesize that Ph-like ALL cells can adapt to JAK kinase inhibition by activating other signaling pathways. To test this hypothesis, we performed a time course experiment in Ph-like ALL cell lines and detected AKT reactivation shortly after JAK-kinase inhibition. In addition, we detected activation of SYK and a reduction in the PI3K negative regulator PTPN6. These observations lead to the hypothesis that Ph-like ALL may have expression and activation of the pre-B cell receptor (pre-BCR). Indeed, gene expression data from the clinical trial P9906 demonstrate that Ph-like ALL cases with CRLF2-rearrangment and JAK2 mutations express high mRNA levels of pre-B cell receptor signaling molecules, including BLNK, IGGL1, VpreB3, and ZAP70. We next hypothesized that pre-BCR mediated activation of PI3 kinase rescues Ph-like ALL cells from JAK inhibition. To study PI3K signaling we first treated Ph-like ALL cell lines and xenografts with idelalisib or INCB-50465, both selective PI3Kδ inhibitors. PI3K inhibition had only a minor effect on cell viability and again AKT was reactivated 48h later. Importantly, combining both JAK-kinase inhibition with PI3K inhibition, prevented AKT reactivation and had strong synergistic effect on cell viability and cell proliferation in vitro. Xenotransplantation models of Ph-like ALL with ruxolitinib and INCB-50465 showed a significantly lower leukemia burden as compared to the single agent treatments but disease eradication was not achieved. Pre-BCR signaling can also activate SRC family kinases and we hypothesized that CRLF2-rearanged Ph-like ALL might be sensitive to SRC inhibition as well. Strikingly, combining JAK2, PI3K, and SRC inhibition further reduced the cell viability by one log scale compared to the JAK2/PI3K inhibitor treated samples and less than 0,2% viable cells were detected, while other ALL subtypes were not affected. In vivo validation of treatment strategy is pending.Clinical relevance: While children with ALL currently have a 5-year survival rate of 90%, adults, especially those over the age of 40, have a very poor clinical outcome with survival rates of about 40%. Adults often do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens. We therefore combined JAK1/2 and PI3K inhibitory treatments with dasatinib, a frequently-used tyrosine kinase inhibitor that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far, the first study using specific signaling inhibitors combined in Ph-like ALL.Conclusion: These findings identify that Ph-like ALL are not oncogene addicted like Ph+ ALL. While BCR-ABL1 activates multiple signaling pathways that can be inhibited with ABL kinase inhibitors, Ph-like ALL need to be treated with multiple inhibitors to induce the same effect. Here we show that a combination of JAK, PI3K and SRC-kinase inhibitors is needed to prevent reactivation of signaling pathways and to induce an apoptotic effect that may be clinically relevant. DisclosuresHurtz:Incyte: Research Funding. Tasian:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Research Funding. Ruggeri:Incyte Corporation: Employment, Equity Ownership. Stubbs:Incyte Corporation: Employment, Equity Ownership. Perl:Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Arog Pharmaceuticals: Consultancy; Novartis: Other: Advisory Board; Seattle Genetics: Other: Advisory board; Astellas: Consultancy; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board. Carroll:Incyte Pharmaceuticals: Research Funding; Astellas Pharmaceuticals: Research Funding.

STAT5BN642H is a driver mutation for T cell neoplasia.

STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.

Inhibitor repurposing reveals ALK, LTK, FGFR, RET and TRK kinases as the targets of AZD1480.

Many tyrosine kinase inhibitors (TKIs) have failed to reach human use due to insufficient activity in clinical trials. However, the failed TKIs may still benefit patients if their other kinase targets are identified by providing treatment focused on syndromes driven by these kinases. Here, we searched for novel targets of AZD1480, an inhibitor of JAK2 kinase that recently failed phase two cancer clinical trials due to a lack of activity. Twenty seven human receptor tyrosine kinases (RTKs) and 153 of their disease-associated mutants were in-cell profiled for activity in the presence of AZD1480 using a newly developed RTK plasmid library. We demonstrate that AZD1480 inhibits ALK, LTK, FGFR1-3, RET and TRKA-C kinases and uncover a physical basis of this specificity. The RTK activity profiling described here facilitates inhibitor repurposing by enabling rapid and efficient identification of novel TKI targets in cells.

B cell-derived IL-4 acts on podocytes to induce proteinuria and foot process effacement.

The efficacy of B cell depletion therapies in diseases such as nephrotic syndrome and rheumatoid arthritis suggests a broader role in B cells in human disease than previously recognized. In some of these diseases, such as the minimal change disease subtype of nephrotic syndrome, pathogenic antibodies and immune complexes are not involved. We hypothesized that B cells, activated in the kidney, might produce cytokines capable of directly inducing cell injury and proteinuria. To directly test our hypothesis, we targeted a model antigen to the kidney glomerulus and showed that transfer of antigen-specific B cells could induce glomerular injury and proteinuria. This effect was mediated by IL-4, as transfer of IL-4-deficient B cells did not induce proteinuria. Overexpression of IL-4 in mice was sufficient to induce kidney injury and proteinuria and could be attenuated by JAK kinase inhibitors. Since IL-4 is a specific activator of STAT6, we analyzed kidney biopsies and demonstrated STAT6 activation in up to 1 of 3 of minimal change disease patients, suggesting IL-4 or IL-13 exposure in these patients. These data suggest that the role of B cells in nephrotic syndrome could be mediated by cytokines.

NVP-BSK805, An Inhibitor of JAK2 Kinase, Significantly Enhances the Radiosensitivity of Esophageal Squamous Cell Carcinoma In Vitro and In Vivo

Esophageal squamous cell carcinoma (ESCC) is a prevalent cancer with high morbidity and mortality in the world. Radiotherapy is one major curative treatment modality for ESCC patients. Our study aimed to find out small-molecular kinase inhibitors that could significantly enhance the sensitivity of radiotherapy in ESCC. Through high-content screening, 93 kinase inhibitors were tested for their radiosensitizing effect on esophageal cancer cells. The radiosensitizing effect of kinase inhibitors was investigated in vitro by clonogenic survival assay, detection of DNA double-strand breaks (DSBs) and by flow cytometry analysis on cell apoptosis. By establishment of xenograft tumor models in BALB/c nude mice, the radiosensitizing effect of kinase inhibitors was investigated in vivo. Our study found that NVP-BSK805, an inhibitor of JAK2 kinase, can significantly radiosensitize ESCC cells by enhancing DSBs and inhibiting DNA damage repair, resulting in decreased clonogenic survival and increased tumor growth inhibitory effect. Mechanism studies discovered that radiation promoted the activation and expression of JAK2 kinase, leading to the development of radioresistance in ESCC cells. JAK2 kinase was highly expressed in tumor tissues of ESCC patients, while rarely expressed in normal esophageal epithelial tissues. Survival analysis revealed JAK2 kinase as a prognostic factor of ESCC patients treated with chmoradiotherapy. Bio-informatics analysis showed that JAK2 kinase regulated those genes that were enriched in DNA recombination and repair pathways. POLQ, one DNA polymerase, was a downstream effector of JAK2 kinase to mediate radioresistance. By whole-exome sequencing (WES) analysis, radiation-induced SNV (single nucleotide variants) in JAK2 kinase was found to be closely associated with the relapse of ESCC patients after radiotherapy. The analysis of RNA sequencing (RNA-seq) showed that IL-1 up-regulated by radiation may be involved in cytokine receptors-mediated JAK2 activation. Our study discovered that NVP-BSK805 could significantly enhance the sensitivity of radiotherapy by inhibiting the expression and activation of JAK2 kinase in ESCC.

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens.

Stimulation of nAchRα7 Receptor Inhibits TNF Synthesis and Secretion in Response to LPS Treatment of Mast Cells by Targeting ERK1/2 and TACE Activation.

The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.

Quantitative Proteome Heterogeneity in Myeloproliferative Neoplasm Subtypes and Association with JAK2 Mutation Status.

Apart from well-known genetic abnormalities, several studies have reported variations in protein expression in Philadelphia-negative myeloproliferative neoplasm (MPN) patients that could contribute toward their clinical phenotype. In this context, a quantitative mass spectrometry proteomics protocol was used to identify differences in the granulocyte proteome with the goal to characterize the pathogenic role of aberrant protein expression in MPNs. LC/MS-MS (LTQ Orbitrap) coupled to iTRAQ labeling showed significant and quantitative differences in protein content among various MPN subtypes [polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)], and according to the genetic status of JAK2 (JAK2V617F presence and JAK2V617F allele burden). A number of differentially expressed proteins were identified, with the most frequent being members of the RAS GTPase family and oxidative stress regulatory proteins. Subsequent analysis found that calreticulin (CALR), known to be involved in calcium homeostasis and apoptotic signaling, was overexpressed in JAK2V617F granulocytes compared with JAK2 wild type and independently of the JAK2V617F allele burden. Finally, it was demonstrated, in a Ba/F3 cell model, that increased calreticulin expression was directly linked to JAK2V617F and could be regulated by JAK2 kinase inhibitors.Implications: In conclusion, these results reveal proteome alterations in MPN granulocytes depending on the phenotype and genotype of patients, highlighting new oncogenic mechanisms associated with JAK2 mutations and overexpression of calreticulin. Mol Cancer Res; 15(7); 852-61. ©2017 AACR.

Nuevas dianas terapéuticas en la enfermedad inflamatoria intestinal

In spite of the best possible optimisation of the available drugs in inflammatory bowel disease (IBD), an important group of patients will require new therapeutic alternatives. Fortunately, there are already new drugs such as vedolizumab or ustekinumab, but, there are many more that will be available in a near future. In this article, a review is made of the evidence and the possible uses of these alternatives. Some of them are drugs that inhibit white cell adhesion, the JAK-kinase inhibitors, the anti-cytokines (especially, ustekinumab), and mongersen.

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans.

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Disorders of the JAK/STAT Pathway in T Cell Lymphoma Pathogenesis: Implications for Immunotherapy

Common gamma receptor-dependent cytokines and their JAK/STAT pathways play pivotal roles in T cell immunity. Abnormal activation of this system was pervasive in diverse T cell malignancies assessed by pSTAT3/pSTAT5 phosphorylation. Activating mutations were described in some but not all cases. JAK1 and STAT3 were required for proliferation and survival of these T cell lines whether or not JAKs or STATs were mutated. Activating JAK and STAT mutations were not sufficient to initiate leukemic cell proliferation but rather only augmented signals from upstream in the cytokine pathway. Activation required the full pathway, including cytokine receptors acting as scaffolds and docking sites for required downstream JAK/STAT proteins. JAK kinase inhibitors have depressed leukemic T cell line proliferation. The insight that JAK/STAT system activation is pervasive in T cell malignancies suggests novel therapeutic approaches that include antibodies to common gamma cytokines, inhibitors of cytokine-receptor interactions, and JAK kinase inhibitors that may revolutionize therapy for T cell malignancies.

Adaptive Reactivation of Signaling Pathways As a Novel Mechanism of Resistance to JAK Inhibitors in Ph-like ALL

Background:Ph-like ALL was first described in 2009 and was accepted as a novel B-ALL subgroup by the WHO in 2016. Ph-like ALL is characterized by different chromosomal rearrangements involving CRLF2, ABL1, JAK2, and EPOR. In addition, mutations in PAX5, IKZF1, and JAK2 are very common. The gene expression profile of this disease is very similar to Ph+ ALL which is characterized by the BCR-ABL1 oncogene. We have previously shown that Ph-like ALL is a high risk disease with a poor prognosis and constitutive activation of cytokine receptor signaling pathway (Tasian et al., submitted). Our current study is focusing on redundant signaling activation in Ph-like ALL that allows the cells to survive cytokine receptor inhibition.Results:We and others have shown that Ph-like ALL have constitutive activation of the cytokine receptor signaling pathway evident by high expression levels of pSTAT5. Xenotransplantation models of Ph-like ALL show a decrease in leukemia burden when treated with Ruxolitinib a JAK1/2 inhibitor. However, all mice ultimately succumb to the disease, demonstrating that Ph-like ALL does not demonstrate oncogene addiction. To understand these results, we have initially used Ph-like ALL cell lines. Ruxolitinib treatment of such cell lines decreases survival but does not eliminate all viable cells. In addition, Ruxolitinib leads to down regulation of pSTAT5, pSTAT3, pAKTT308 as well as pAKTS473. To further study the mechanism of resistance to JAK inhibition we performed a time course experiments and found that as early as 48-72 hours after the initial JAK inhibitor treatment Ph-like ALL cell are able to reactivate pSTAT5 and pAKT. This effect was not due to drug inactivation. Based on this observation, we hypothesized that adaptive signaling through PI3 kinase protects Ph-like ALL cells from cell death. Interestingly, PI3K inhibition (Idelalisib) alone had only a minor effect on cell viability and cell proliferation and the Ph-like ALL cells were able to reactivate the signaling network within 48-72h. Dual treatment with JAK1/2 and PI3 kinase inhibition significantly increased the apoptotic effect and stopped proliferation. In addition, the dual treatment induced a long lasting inhibitory effect with no signs of pAKT reactivation. Strikingly, combining JAK and PI3K inhibition with Methotrexate an antimetabolite that is used for the treatment of ALL patients significantly increased the apoptotic effect of the combinatory treatment. Overall, these results demonstrate that adaptive signaling with reactivation of the PI3K signaling cascade protects Ph-like ALL cells from inhibition of JAK kinases and suggests that dual kinase inhibitor therapy may increase response in the clinic. Experiments studying how reactivation occurs and the in vivo response to dual JAK-PI3K inhibition are in progress.Clinical relevance:Older patients (>40 years) often do not qualify for induction chemotherapy as they do not tolerate high doses of chemotherapy regimens and are therefore in clinical need of alternative treatment regimens that can improve their clinical outcome. While children with ALL currently have a 5-year survival rate of 90% adults especially older adults have a very poor clinical outcome with survival rates of about 40%. We therefore combined JAK1/2 and PI3K inhibitory treatments with methotrexate a frequently used antimetabolite that is well tolerated by older patients. Strikingly, the combinatory treatment had a strong effect on cell viability and represents so far the first study using specific signaling inhibitors combined with an antimetabolite treatment in Ph-like ALL.Conclusion:These findings identify that Ph-like ALL are able to reestablish their signaling networks. Based on our finding it is important to target more than one signaling pathways to fully inhibit cell proliferation and cell survival. We propose to treat Ph-like ALL patients with a combinatory treatment of JAK1/2 and PI3K inhibitor and currently used antimetabolite drugs to reduce the risk of relapse and to improve the clinical outcome of patients with Ph-like ALL. DisclosuresPerl:Seattle Genetics: Consultancy; Actinium Pharmaceuticals, Inc.: Research Funding.

Selective Inhibition of Nuclear Cytoplasmic Transport As a New Treatment Paradigm in Myelofibrosis

▪Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by constitutive activation of JAK/STAT signaling due to mutations in JAK2, calreticulin or MPL. Many MF patients suffer from severe constitutional symptoms and have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib (RUX) reduce MF symptoms, but like all other drugs used in MF, are not curative, with persistence of mutant cells and prompt symptom rebound upon discontinuation. This defines a clinical need to identify strategies capable of inducing more profound and durable responses in MF.To identify previously unrecognized molecular vulnerabilities in MF, we infected HEL cells (homozygous for JAK2V617F) with a barcoded lentiviral shRNA library targeting ~5,000 human signal transduction genes, with 5-6 shRNAs/gene (Cellecta Human Module 1). Conditions were optimized to achieve a multitude of infection (MOI) of ~1. Barcode abundance was compared between days 0 and 9 after infection by next generation sequencing. Candidates were selected based on ≥ 15-fold reduction of abundance by ≥ 2 shRNAs targeting the same gene, similar to Khorashad et al. [Blood. 2015;125(11):1772-81]. Amongst the genes meeting these criteria, nuclear cytoplasmic transport (NCT) was significantly enriched, with RAN and RANBP2 amongst the top genes, suggesting that HEL cells may be highly dependent on NCT. For confirmation, HEL cells were stably transduced with doxycycline (DOX)-inducible shRAN. After 72 hours DOX-induced knockdown of RAN reduced viable cells by 77.3±5.5% and colony formation by 82.8±1.3% and dramatically increased apoptosis (uninduced: ~10% vs. induced: ~50%). Similar results were observed in SET-2 cells (heterozygous for JAK2V617F). We next cultured HEL and SET-2 cells with graded concentrations of the KPT-330 (selinexor, Karyopharm), an inhibitor of CRM-1, the core component of NCT, or RUX as a comparison. Selinexor was five-fold more potent than RUX against HEL cells (IC50: 98nM for KPT vs. 536 nM for RUX) and as potent as RUX in SET-2 cells (IC50:~100 nM). Importantly, RUX-resistant HEL cells (IC50:24µM) were highly sensitive to inhibition of NCT by knockdown of RAN or selinexor (IC50:160nM). Selinexor also selectively inhibited colony formation by primary MF vs. cord blood (CB) CD34+ cells (IC50:93nM for MF vs. 203nM for CB). Lastly, selinexor enhanced RUX-induced growth inhibition and apoptosis in primary MF CD34+cells cultured ex vivo for 72h (including both JAK2 mutation positive and negative MF samples, n=3 for each, and RUX resistant patient samples, n=6). Nuclear:cytoplasmic fractionation of HEL cells revealed that the expression and nuclear localization of the tumor suppressors FoxO3A and APC, but not of PP2A and nucleophosmin (NPM) were significantly increased upon knockdown of RAN, which may contribute to the increased apoptosis following NCT inhibition.To determine the in vivo effects of selinexor in MF, we induced MPN in Balb/c mice by transplanting donor marrow infected with JAK2V617F for three weeks, and then treated mice (n=13/group) with vehicle, selinexor (initial dose 20 mg/kg, 3x weekly, orally) or RUX (initial dose 50 mg/kg twice daily, orally) or the combination of RUX plus selinexor for up to 4 weeks. Combination treatment significantly reduced white blood cell counts and normalized spleen size. Compared to vehicle, selinexor alone significantly reduced GFP+cells in the spleen, and this effect was further enhanced with the combination treatment. Histopathology revealed that combination treatment restored splenic architecture, while bone marrow fibrosis was not significantly altered by selinexor or the combination. Mice in all groups, including the combined vehicle controls, experienced considerable weight loss, suggesting that toxicity may be partially due to high dose and frequent drug administration. Experiments with the next generation NCT inhibitor KPT-8602 [Etchin et al., Leukemia, 2016 Jun 24] are underway.In summary, our results suggest that MF cells are exquisitely dependent on NCT, and that NCT inhibition alone or in combination with RUX may reduce JAK2V617F allelic burden. This identifies NCT as a prime therapeutic target in MF. A phase I clinical trial of selinexor in refractory MF is in preparation. DisclosuresBaloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Deininger:BMS: Consultancy, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

HSP27: A Therapeutic Target in Myelofibrosis

▪Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Despite their beneficial effects on spleen size and symptoms, JAK2 inhibitors induce low molecular and survival responses underscoring the urgent need for other therapeutic approaches. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins like HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary, lung or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated in this work, the status of HSP27 in MF patient's samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427.In this study, we first assessed the extracellular and intracellular level of HSPs from MF patients by ELISA, flow cytometry and by immunohistochemistry. We observed for the first time a specific increase in both intracellular and extracellular HSP27 in CD34+ circulating hematopoietic progenitor cells (n=9-16; P=0.0097) and in the sera of patients (n=24-27; P<0.0001) with MF compared with healthy donors, respectively. Moreover, we identified the presence of HSP27 in the bone marrow’s MF patients.We then investigated the in vivo impact of OGX-427, a specific inhibitor of HSP27, or an oligonucleotide control in a murine TPO-induced MF mouse model. The use of OGX-427 limited the progression of the disease in our MF mouse model (n=9). In particular, OGX-427 was associated with a marked reduction in both the spleen weight and size. Also, we noted a decrease of megakaryocyte hyperplasia in the bone marrow accompanied by a visible restoration of spleen structure and lymphoid white pulp territories with OGX-427.Taking altogether, our results support a key role of HSP27 in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder. DisclosuresNo relevant conflicts of interest to declare.

CSF3R Mutations Are Predominantly Subclonal Events in Intermediate Risk Karyotype AML and Prevalently Occur with CEBPA Mutations

Background: The colony stimulating factor 3 receptor(CSF3R) plays an important role in granulocyte differentiation and acquired mutations have been described in different myeloid malignancies. Besides chronic neutrophilic leukemia (CNL) and severe congenital neutropenia (SCN), mutations in CSF3R (CSF3Rmut) have also been reported in acute myeloid leukemia (AML) at low frequency and recently have been associated with CEBPA mutations (Lavallèe et al., Blood 2016; Maxson et al., Blood 2016) in adult and pediatric AML. First studies suggest sensitivity of CSF3Rmut CNL against JAK kinase inhibitors suggesting the possibility for targeted therapy for a subset of CSF3Rmut AML patients.Aims: The evaluation of CSF3Rmut in AML with intermediate-risk karyotype for frequency and association with other mutations with special focus on CEBPA mutation status.Methods: We analyzed 274 cases with intermediate risk de novo AML for CSF3Rmut by next generation sequencing (Illumina, San Diego, CA) of exon 14 and 17 (sensitivity: 1%), which contain the mutational hotspot regions of CSF3R. As CSF3R mutations were associated with CEBPA mutations in the above-mentioned studies, we analyzed a selected cohort of 179 CEBPA mutated cases in comparison to 95 cases with CEBPA wild type.202 cases (74%) had normal karyotype (CN-AML) and 72 (26%) had intermediate-risk aberrant cytogenetics according to MRC criteria. Female/male ratio was 127/147 and age ranged from 16- 88 y (median: 64 y). Mutation status of the following genes were available in all cases: ASXL1, CEBPA, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, KMT2A-PTD, NPM1, NRAS, RUNX1 and WT1. All mutations with a variant allele frequency (VAF) < 10% were defined as subclonal. 95 cases had wild type CEBPA (CEBPAwt), 92 cases were CEBPA single mutated (CEBPAsm), 81 cases were CEBPA double mutated (CEBPAdm), 6 cases showed CEBPA mutations withloss of heterozygosity (CEBPA LOH).Results: Overall, in 7/274 patients (3%) CSF3Rmut were detected. This is slightly higher than expected in an unselected AML cohort (Sano et al., Br J Haematol 2015). Cytomorphology revealed AML M0 (n=1), AML M1 (n=2), AML M2 (n=3) and AML M4 (n=1). With regard to immunophenotype CSF3Rmut cases did not differ from CSF3Rwt cases which may be due to limited case numbers. All CSF3Rmut cases had CN-AML. The activating transmembrane mutation p.Thr618Ile, which has been identified as common mutation in CNL was the most frequent mutation (4/7). In addition two mutations leading to a truncated receptor (p.Ser715* and p.Gln749*) and a previously not described frame-shift mutation leading to a defecting splice variant (p.Ser783Glnfs*6) were identified. The median CSF3R VAF was 7% (range 2-45%). In 5/7 cases, CSF3Rmut was subclonal (VAF <10%, see figure). CSF3Rmut cases showed a median of 2 concomitant mutations (range 1-4). The most frequent concomitantly mutated gene was CEBPA (5/7 cases; 2 cases CEBPAsm, 1 case CEBPAdm and 2 cases with CEBPA LOH), followed by NRAS (3/7), ASXL1 (2/7) and RUNX1 (2/7). IDH1, TET2 and WT1 were mutated in one case each (see figure). Mutations in NPM1, FLT3, KMT2A or GATA2 were never detected. Interestingly, in CEBPAdm and CEBPA LOH cases, CSF3R was the only concomitantly mutated gene, whereas CEBPAsm cases harbored mutations in at least 1 gene in addition to CSF3R. In 2/7 CSF3Rmut cases we analyzed changes in the patterns of genetic lesions between diagnosis and relapse. Case 1 harbored CEBPAdm and a subclonal CSF3Rmut (VAF: 7%) at diagnosis. At relapse the CEBPAdm persisted, whereas CSF3Rmut was lost. Case 2 harbored two germline CEBPAmut and one somatic CEBPAmut at diagnosis. In addition, mutations in CSF3R and WT1 were identified. At relapse both the somatic CEBPAmut and WT1mut persisted, whereas CSF3Rmut was lost. These results indicate again that the CSF3Rmut was present only in a subclone of CEBPAmut AML in both cases.Due to the limited number of CSF3Rmut cases, we did not perform survival analysis in this study.Summary: 1) CSF3R mutations are rare and predominantly subclonal events in AML and therefore may not be a suitable target for directed therapy.2) CSF3Rmut prevalently occur with CEBPA mutations but are not limited to CEBPAdm. [Display omitted] DisclosuresFasan:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.