Deletion Of Jak2 Research Articles

Clinicopathologic characteristics of myeloproliferative neoplasms with JAK2 exon 12 mutation

The presence of JAK2 exon 12 mutation was included by the 2016 World Health Organization (WHO) Classification as one of the major criteria for diagnosing polycythemia vera (PV). Few studies have evaluated the clinical presentation and bone marrow morphology of these patients and it is unclear if these patients fulfill the newly published criteria of 5th edition WHO or The International Consensus Classification (ICC) criteria for PV. Forty-three patients with JAK2 exon 12 mutations were identified from the files of 7 large academic institutions. Twenty patients had complete CBC and BM data at disease onset. Fourteen patients met the diagnostic criteria for PV and the remaining six patients were diagnosed as MPN-U. At diagnosis, 9/14 patients had normal WBC and platelet counts (isolated erythrocytosis/IE subset); while 5/14 had elevated WBC and/or platelets (polycythemic /P subset). We found that hemoglobin and hematocrit tended to be lower in the polycythemia group. Regardless of presentation (P vs IE), JAK2 deletion commonly occurred in amino acids 541–544 (62 %). MPN-U patients carried JAK2 exon 12 mutation, but did not fulfill the criteria for PV. Half of the patients had hemoglobin/hematocrit below the diagnostic threshold for PV, but showed increased red blood cell count with low mean corpuscular volume (56–60 fL). Three cases lacked evidence of bone marrow hypercellularity. In summary, the future diagnostic criteria for PV may require a modification to account for the variant CBC and BM findings in some patients with JAK2 exon 12 mutation.

JAK2-CHK2 signaling safeguards the integrity of the mitotic spindle assembly checkpoint and genome stability

Checkpoint kinase 2 (CHK2) plays an important role in safeguarding the mitotic progression, specifically the spindle assembly, though the mechanism of regulation remains poorly understood. Here, we identified a novel mitotic phosphorylation site on CHK2 Tyr156, and its responsible kinase JAK2. Expression of a phospho-deficient mutant CHK2 Y156F or treatment with JAK2 inhibitor IV compromised mitotic spindle assembly, leading to genome instability. In contrast, a phospho-mimicking mutant CHK2 Y156E restored mitotic normalcy in JAK2-inhibited cells. Mechanistically, we show that this phosphorylation is required for CHK2 interaction with and phosphorylation of the spindle assembly checkpoint (SAC) kinase Mps1, and failure of which results in impaired Mps1 kinetochore localization and defective SAC. Concordantly, analysis of clinical cancer datasets revealed that deletion of JAK2 is associated with increased genome alteration; and alteration in CHEK2 and JAK2 is linked to preferential deletion or amplification of cancer-related genes. Thus, our findings not only reveal a novel JAK2-CHK2 signaling axis that maintains genome integrity through SAC but also highlight the potential impact on genomic stability with clinical JAK2 inhibition.

Specific Ablation of Jak2 from CD11c+ Cells Attenuates Salt‐Sensitive Hypertension through an ENaC‐Dependent Mechanism

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase‐dependent formation of immunogenic isolevuglandin (IsoLG)‐protein adducts leading to inflammation and salt‐sensitive hypertension. We hypothesized that expression of Jak2 specifically in antigen presenting myeloid cells contributes to salt‐sensitive hypertension in an ENaC dependent mechanism. Profiling whole transcriptome using RNA‐Seq and analyzing the gene differential expression patterns in human monocytes reveals that high salt treatment upregulates genes of the Jak/STAT pathway, and the downstream regulators suppressor of cytokine signaling (SOCS) genes. Male and female mice lacking Jak2 in CD11c+ cells developed blunted hypertension (123.8±4.7) during the high salt feeding phase of the N‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)/high salt model of salt‐sensitive hypertension compared to the wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention in response to high salt‐feeding when compared to the wildtype littermate controls. We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ, and serum/glucocorticoid regulated kinase 1 (SGK1) in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less renal infiltration of effector memory T cells (TEM), less aortic infiltration of CD11c+ cells with less expression of CD86, and less production of IsoLGs and IL1‐beta. These results indicate that dendritic cell Jak2 plays an important role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.

IL-1 Signaling Promotes Clonal Expansion and Progression of Bone Marrow Fibrosis in JAK2V617F-Induced Myeloproliferative Neoplasm

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell derived myeloid malignancies characterized by aberrant production of myeloid, erythroid or megakaryocytic lineage cells. JAK2V617F is the most common somatic driver mutation associated with MPN. Interestingly, JAK2V617F mutation can also be detected in healthy individuals with clonal hematopoiesis of indeterminate potential (CHIP) who do not exhibit overt changes in blood counts. This suggests that other factors might be involved in association with JAK2 mutation in clonal expansion and initiation/progression of MPN. Chronic inflammation is frequently associated with MPN. Interleukin 1 (IL-1) is a major regulator of inflammation. IL-1 consists of two related cytokines IL-1α and IL-1β. Both IL-1α and IL-1β bind to the IL-1 receptor 1 (IL-1R1) to initiate downstream signaling. Although elevated expression of IL-1α and IL-1β has been observed in MPN, their role in the pathogenesis of MPN has remained elusive.In this study, we investigated the role of IL-1 signaling in JAK2V617F-induced MPN using a Jak2V617F knock-in mouse model. We observed elevated levels of IL-1α and IL-1β in mice expressing heterozygous (Jak2 VF/+) and homozygous Jak2V617F (Jak2 VF/VF) compared with WT control animals. Notably, IL-1α and IL-1β expression was significantly higher in Jak2 VF/VF mice exhibiting extensive bone marrow (BM) fibrosis compared with Jak2 VF/+ mice exhibiting polycythemia vera (PV), consistent with elevated levels of IL-1 in patients with myelofibrosis (MF).Since both IL-1α and IL-1β levels were elevated in Jak2 VF/VF mice exhibiting MF, we utilized conditional IL-1R1 knockout (IL-1R1cKO) and Jak2 VF/VF mice to assess the role of IL-1 signaling in the initiation/progression of MF. As expected, Jak2 VF/VF mice exhibited a significant increase in WBC, neutrophil and platelet counts compared to WT control mice. Deletion of IL-1R1in Jak2 VF/VF mice (IL-1R1cKO; Jak2 VF/VF) significantly reduced the WBC, neutrophil and platelet counts to almost control levels. Flow cytometric analysis also showed a significant reduction of myeloid (Gr-1 +) and megakaryocytic (CD41 +) precursors in the BM and spleens of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Moreover, deletion of IL-1R1 significantly reduced hematopoietic stem and progenitor cells (HSPC) in the BM of IL-1R1cKO; Jak2 VF/VF mice compared to Jak2 VF/VF mice. Spleen weight was significantly reduced in IL-1R1cKO; Jak2 VF/VF mice compared with Jak2 VF/VF mice and they were comparable to control WT mice. More importantly, deletion of IL-1R1 markedly reduced BM fibrosis in Jak2 VF/VF mice. These data suggest an important role of IL-1 signaling in the progression of BM fibrosis in Jak2V617F-induced MPN.To test whether IL-1 signaling contributes to clonal expansion of JAK2 mutant HSPC, we performed competitive transplantation assays by mixing Mx1Cre; Jak2 VF/+ and Mx1Cre; IL-1R1 F/F; Jak2 VF/+ mice BM cells with CD45.1 + WT mice BM cells at a ratio of 1:1 and transplanted into lethally irradiated CD45.1 + recipient animals. At 4 weeks after BMT, the recipient animals were injected with pI-pC to induce Jak2V617F expression and IL-1R1 deletion. We observed significantly higher percentages of total CD45.2 + cells as well as CD45.2 + myeloid (Gr-1 +), B- and T-cells in the peripheral blood of chimeric mice receiving Jak2 VF/+ BM compared with chimeric mice receiving IL-1R1cKO; Jak2 VF/+ BM. We also observed significantly reduced percentages of CD45.2 + LSK, LK, Gr-1 + and CD41 + cells in the BM of chimeric recipient animals receiving IL-1R1cKO; Jak2 VF/+ BM compared with Jak2 VF/+ BM. These results suggest a role of IL-1 signaling in clonal expansion of Jak2V617F mutant HSPC.Additionally, we tested the effects of blocking IL-1R1 using an anti-IL-1R1 antibody in the homozygous Jak2V617F knock-in mouse model of MF. We observed that anti-IL-1R1 antibody treatment significantly reduced peripheral blood WBC and neutrophil counts and decreased HSPC and myeloid precursors in the BM of Jak2 VF/VF mice. Furthermore, anti-IL-1R1 antibody treatment significantly reduced splenomegaly and markedly reduced BM fibrosis in Jak2 VF/VF mice, suggesting that therapies targeting IL-1R1 could be useful for the treatment of myelofibrosis. Overall, our results suggest that IL-1 signaling contributes to clonal expansion of Jak2V617F mutant HSPC and progression of bone marrow fibrosis in MPN. DisclosuresNo relevant conflicts of interest to declare.

Bleeding diathesis in mice lacking JAK2 in platelets

AbstractThe tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells, such as hematopoietic stem cells and megakaryocytes (MKs). Individuals expressing the somatic JAK2 V617F mutation commonly develop myeloproliferative neoplasms (MPNs) associated with venous and arterial thrombosis, a leading cause of mortality. The role of JAK2 in hemostasis remains unclear. We investigated the role of JAK2 in platelet hemostatic function using Jak2fl/fl Pf4-Cre (Jak2Plt−/−) mice lacking JAK2 in platelets and MKs. Jak2Plt−/− mice developed MK hyperplasia and splenomegaly associated with severe thrombocytosis and bleeding. This notion was supported by failure to occlude in a ferric chloride carotid artery injury model and by a cremaster muscle laser-induced injury assay, in which Jak2Plt−/− platelets failed to form stable thrombi. Jak2Plt−/− platelets formed thrombi poorly after adhesion to type 1 collagen under arterial shear rates. Jak2Plt−/− platelets spread poorly on collagen under static conditions or on fibrinogen in response to the collagen receptor GPVI-specific agonist, collagen-related peptide (CRP). After activation with collagen, CRP, or the CLEC-2 agonist rhodocytin, Jak2Plt−/− platelets displayed decreased α-granule secretion and integrin αIIbβ3 activation or aggregation, but showed normal responses to thrombin. Jak2Plt−/− platelets had impaired intracellular signaling when activated via GPVI, as assessed by tyrosine phosphorylation. Together, the results show that JAK2 deletion impairs platelet immunoreceptor tyrosine-based activation motif signaling and hemostatic function in mice and suggest that aberrant JAK2 signaling in patients with MPNs affects GPVI signaling, leading to hemostatic platelet function.

Palbociclib in advanced acral melanoma with genetic aberrations in the cyclin-dependent kinase 4 pathway

BackgroundGenetic aberrations in the cyclin-dependent kinase (CDK)4 pathway occur in 82% of patients with acral melanoma (AM), which is the predominant subtype of melanoma in China. We aimed to evaluate the anti-tumour activity of palbociclib, a selective CDK4/6 inhibitor, in patients with advanced AM with CDK4 pathway gene aberrations. MethodsIn this phase II trial, patients with advanced AM with CDK4 or/and CCND1 gain or/and CDKN2A loss were treated with oral palbociclib (125 mg) on days 1–21 of a 28-day cycle. The primary end-point was overall response rate (ORR). Secondary end-points were progression-free survival (PFS), overall survival (OS), and treatment-related adverse events (TRAEs). Whole-exome sequencing and multiplex immunohistochemistry of the available formalin-fixed, paraffin-embedded samples of nine patients were analysed to explore the predictive biomarkers of palbociclib response. ResultsFifteen patients were enrolled. Three (20.0%) patients achieved tumour shrinkage at 8 weeks, including one with confirmed partial response. At data cut-off date, treatment was ongoing for one patient. The median PFS was 2.2 mo (range: 1.5–13.3 mo; 95% confidence interval [CI]: 1.9–2.5), and the median OS was 9.5 mo (range: 2.6–14.1 mo, 95% CI: 5.7–13.4). Eight patients died due to disease progression. The most common TRAEs were leukopenia (87%; Grade III/IV, 27%), neutropenia (80%; grade III/IV, 27%), and fatigue (53%; grade III/IV, 7%). Significant JAK2 deletions and SH2B3 amplifications were observed in patients who did not achieve any clinical benefit (CB) with palbociclib treatment. MCM7 amplification or protein expression level was found to be associated with CB. ConclusionsPalbociclib monotherapy demonstrated preliminary efficacy and an acceptable safety profile in advanced AM patients with CDK4 pathway aberrations. Patients with amplification or high protein levels of MCM7 were more prone to benefit from palbociclib. The JAK-STAT pathway might play a role in the mechanism of action of palbociclib in AM. Trial registration numberNCT03454919. The date of registrationMarch 6, 2018.

Bleeding Diathesis in Mice Lacking JAK2 in Platelets

The tyrosine kinase JAK2 is a critical component of intracellular JAK/STAT cytokine signaling cascades that is prevalent in hematopoietic cells such as hematopoietic stem and progenitor cells (HSPCs), megakaryocytes (MKs), and platelets. Individuals expressing somatic activating JAK2 mutations such as JAK2V617F commonly develop myeloproliferative neoplasms (MPNs) associated with serious complications, including venous and arterial thrombosis, a leading cause of mortality. Here, we investigated the role of JAK2 in hemostasis and thrombosis using Jak2fl/flPf4-Cre (Jak2Plt-/-) mice specifically lacking JAK2 within the platelet lineage. Jak2Plt-/- mice developed severe thrombocytosis with a 5-fold increase in circulating platelet number, MK hyperplasia, and splenomegaly. Jak2Plt-/-platelets were of normal size and the expression of major membrane surface glycoproteins was indistinguishable from controls, except for the integrin β3, which was reduced by 20%. Despite the thrombocytosis, Jak2Plt-/- mice had a severe bleeding diathesis, as evidenced by: 1) prolonged tail bleeding time; 2) failure to occlude in a ferric chloride-induced carotid artery injury model; and 3) failure to form stable thrombi in a laser-induced cremaster muscle injury model. Jak2Plt-/- platelets spread poorly on immobilized collagen or on immobilized fibrinogen following GPVI stimulation with the collagen-related peptide (CRP). Jak2Plt-/- platelets had defective α-granule secretion and integrin αIIbβ3 activation following stimulation with CRP, but not thrombin, and showed aggregation defects with low-doses of CRP. Together, the data support a GPVI-specific impairment in platelets lacking JAK2, a notion that was supported by impaired intracellular signaling following GPVI stimulation, as assessed by protein tyrosine phosphorylation. Jak2Plt-/- platelets adhered poorly to type I collagen under arterial shear rates in whole blood. However, JAK2 deletion in platelets did not alter plasma von Willebrand factor (VWF) levels or botrocetin-mediated binding of plasma VWF to GPIbα. Together, the results underline a critical role for JAK2 in platelet GPVI signaling and hemostatic function, which likely contributes to venous and arterial thrombosis observed in patients with MPNs with the activating JAK2V617F mutation. Disclosures No relevant conflicts of interest to declare.

Abstract P131: Jak2 Expression In Cd11c+ Myeloid Cells Plays A Role In Salt-sensitive Hypertension Through An Enac-dependent Mechanism.

Hypertension is a major risk factor for development of cardiovascular disease. Excess dietary salt contributes to inflammation and the genesis of hypertension. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and salt-sensitive hypertension. We hypothesized that Jak2 expression, specifically in CD11c + myeloid cells, regulates expression of ENaCγ and promotes salt-sensitive hypertension. Our results indicate that deletion of Jak2 in CD11c + myeloid cells reduced the salt-induced expression of ENaCγ in CD11c+ cells. Moreover, mice lacking Jak2 in CD11c+ cells developed a blunted hypertensive response (123.8±4.7) during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L- NAME)/high salt model of salt-sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt-feeding when compared to the wildtype litter mate controls (57.89±9.5). These results indicate that Jak2 expression in CD11c + myeloid cells plays a role in salt- sensitive hypertension through an ENaC-dependent mechanism.

Dendritic Cell Specific Jak2 Expression plays a role in in Salt‐Sensitive Hypertension through an ENaC‐dependent Mechanism

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase‐dependent formation of immunogenic isolevuglandin (IsoLG)‐protein adducts leading to inflammation and salt‐sensitive hypertension. We hypothesized that Jak2 expression in dendritic cells regulates expression of ENaCγ and promotes salt‐sensitive hypertension. Mice lacking Jak2 in CD11c+ cells developed blunted hypertension (123.8±4.7) during the high salt feeding phase of the N‐Nitro‐L‐arginine methyl ester hydrochloride (LNAME)/high salt model of salt‐sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt‐feeding when compared to the wildtype litter mate controls (57.89±9.5). We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less aortic infiltration of CD11c+ cells with less expression of CD86 and production of IsoLGs. These results indicate that dendritic cell Jak2 plays a role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.Support or Funding InformationK01HL130497R01HL147818T32HL144446

Adipocyte JAK2 mediates spontaneous metabolic liver disease and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.

PS1020 SNP ARRAY REVEALS A NEW DELETION OF JAK2 IN AML PATIENTS

Background:Acute Myeloid Leukemia (AML) is a disease of the elderly that initiates in a hematopoietic stem cell and is characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background.Aims:In Acute Myeloid Leukemia (AML) there is a strong need to develop new diagnostic and therapeutic options: to identify the genes mostly predictive of treatment response, we use Single Nucleotide Polymorphism (SNP) Arrays and Whole Exome Sequencing (WES) in AML patients with heterogeneous karyotypes and different subgroups.Methods:SNP arrays (CytoScan HD Array, Affymetrix Inc.) was positively done in 58 AML samples and all of them were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 software (BioDiscovery), while Next Generation Sequencing (NGS)‐WES HiSeq 2000 (Illumina) was done in 35 AML patients.Results:We treated 58 AML patients (pts) with a median age of 52 years, of which 9 were insensitive pts and 49 sensitive to therapies, all obtaining complete clinical remission. Copy Number Alterations (CNAs) were detected in all patients affecting all the chromosomes, in particular our cohort of pts present a percentage of CNA, divided as follow: 58% of LOH, 15% of gain, 16% of loss, 3% of high copy gain and 4% of homozygous copy loss. We found that several cancer genes were preferentially amplified: IGH@, KIT, IGL@, TSC1, NOTCH2, SETD2, EZH2, while in high copy gain we found TSC1, PTEN, RB1, IKZF1, ZRSR2, IGH@, NF1, MYC, KRAS. Then several genes were preferentially deleted: CRLF2, ATRX, JAK2, BCOR, PHF6, GATA1, KDM6A and in homozygous copy loss: JAK2, CRLF2, RB1, PDGFRA, RUNX1. Moreover we genes in LOH: DDX5, MTCP1, HOOK3, ZRSR2, GATA1, KDM6A, BCOR, NF1, BRAF, ATRX. We focused on two losses of JAK2: the first deletion, detected in 18/58 (31%) pts, goes from 5030 to 5038 Kbp (7,43 Kbps) including intron 4–5; the second minimal common region of loss, detected in 5/58 (8,6%) pts, goes from 5083 to 5098 Kpb (15 Kbps) including intron 19–20 and exons 20, 21 and 22, suggesting a defective transduction, in fact we showed that the overall survival rate is better for the group of pts which present a deletion of JAK2 rather than the group with an amplification of this gene (p‐value <0,01). We have also found three other genes which are preferentially lost: SIRPB1, ADAM3A and STAG2 with a percentage of 50%, 43% and 69% respectively.By NGS‐WES we analyzed 35 AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of response to treatment. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR.Summary/Conclusion:By SNP arrays we have identified Copy Number Alterations involving important cancer genes AML and we showed that a new deletion in JAK2 may have a role in overall survival rate. Future prospective will be to correlate the cancer genes alteration and mutations with the prognosis of AML, in order to identify new biomarkers relevant for the disease.

Loss of JAK1 Drives Innate Immune Deficiency.

The Janus kinase—signal transducers and activators of transcription (JAK-STAT) signaling pathway is critical in tuning immune responses and its dysregulation is tightly associated with cancer and immune disorders. Disruption of interleukin (IL)-15/STAT5 signaling pathway due to the loss of IL-15 receptor chains, JAK3 or STAT5 leads to immune deficiencies with natural killer (NK) cell abnormalities. JAK1, together with JAK3 transmits signals downstream of IL-15, but the exact contribution of JAK1 to NK cell biology remains to be elucidated. To study the consequences of JAK1 deficiency in NK cells, we generated mice with conditional deletion of JAK1 in NKp46+ cells (Jak1fl/flNcr1Cre). We show here that deletion of NK cell-intrinsic JAK1 significantly reduced NK cell numbers in the bone marrow and impaired their development. In line, we observed almost a complete loss of NK cells in the spleen, blood, and liver, proving a crucial role of JAK1 in peripheral NK cells. In line, Jak1fl/+Ncr1Cre mice showed significantly impaired NK cell-mediated tumor surveillance. Our data suggest that JAK2 is not able to compensate for the loss of JAK1 in NK cells. Importantly, conditional deletion of JAK2 in NKp46+ cells had no effect on peripheral NK cells revealing that NK cell-intrinsic JAK2 is dispensable for NK cell survival. In summary, we identified that loss of JAK1 in NK cells drives innate immune deficiency, whereas JAK2 deficiency leaves NK cell numbers and maturation unaltered. We thus propose that in contrast to currently used JAK1/JAK2 inhibitors, the use of JAK2-specific inhibitors would be advantageous for the patients by leaving NK cells intact.

PO-399 Tumour CDKN2A loss predisposes to immunotherapy resistance

IntroductionIn recent years, treatment with immunomodulating antibodies targeting the inhibitory T-cell receptor PD-1 led to durable clinical responses in a subgroup of patients with melanoma, lung cancer or bladder cancer. Nevertheless, the majority of the patients do not respond, and the mechanisms underlying primary or acquired resistance are still poorly defined. Recently, it was demonstrated that mutations in the JAK1/2-STAT1 signalling pathway play an essential role in protecting tumour cells from the anti-proliferative and pro-apoptotic activity of T cell-derived IFNg thereby contributing to immunotherapy resistance.Material and methodsJAK2 mutational status in melanoma cells was defined by targeted sequencing. JAK2 and p16 expression was studied by immunoblotting. Single nucleotide polymorphism array data of 46 melanoma cell lines was screened for deletions on Chr.9p to which JAK2 and CDKN2A (encoding p16) map. cBioPortal for Cancer Genomics was applied to analyse melanoma, lung squamous cell carcinoma and bladder urothelial carcinoma data sets of The Cancer Genome Atlas (TCGA) for association of CDKN2A and JAK2 deletions. The UCSC Xena Browser was used to visualise alterations in Chr.9p segments of TCGA tissue samples.Results and discussionsIn this study we demonstrate the genetic evolution of JAK2 deficiency in melanoma cells. JAK2-deficient melanoma cells showed an inactivating mutation in one JAK2 allele and loss of the second allele. SNP array analyses revealed large deletions on chr9p in these cells, in the region where JAK2 maps together with CDKN2A, the latter encoding the tumour suppressor p16. Loss of CDKN2A is known as an early frequent event in melanoma development. Accordingly, in a cohort of 46 melanoma cell lines, we detected CDKN2A-associated JAK2 deletions in more than 75%. Co-deletion of JAK2 and CDKN2A was detected also in TCGA melanoma tissues and, interestingly, in a large fraction of samples from different tumour types, including lung squamous cell carcinoma and bladder urothelial carcinoma.ConclusionThis result demonstrates that tumours harbouring allelic CDKN2A deletions may be more prone to develop resistance to immunotherapy due to associated JAK2 allele losses.

Tumor CDKN2A-Associated JAK2 Loss and Susceptibility to Immunotherapy Resistance

Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy.

Abstract 585: Chromosome 9p24.1 deletions as a determinant of tumor immune surveillance and immune checkpoint blockade therapy in non-small cell lung cancer

Abstract Anti-PD-1/PD-L1 immune checkpoint blockade therapy requires the presence of PD-L1/PD-L2 and MHC class I antigens on the tumor cell surface. We previously found JAK1 truncating mutations in endometrial cancer cells that impairs JAK1/JAK2-STAT1-IRF1-mediated signaling pathway, which regulates MHC class I antigen presentation. To determine the potential role and genetic defects in the IFN-γ-IRF1 pathway in non-small cell lung cancer (NSCLC), we analyzed the Cancer Genome Atlas (TCGA) data. Expression of IRF1 correlated with cytolytic activity markers GZMA and PRF1. Loss-of-function (LOF) genetic alterations in the IFN-γ-IRF1 pathway genes were found in 64 cases (6.3%) among 1016 patients. These genetic defects occurred prevalently in the JAK2 gene (33 cases) and often through deletions (29 cases) of chromosome 9p24.1. JAK2 gene deletions were frequently associated with deletions of CD274 and PDCD1LG2 genes that encode PD-L1 and PD-L2, respectively, whereas CD274 and PDCD1LG2 deletions were always accompanied by JAK2 deletions. Chromosome 9p is frequently deleted in NSCLC. This has been attributed to CDKN2A/CDKN2B and PTPRD tumor suppressor genes located at the chromosome 9p21.3-9p24.1. Interestingly, JAK2, CD274, and PDCD1LG2 genes are co-localized at chromosome 9p24.1 near the CDKN2A/CDKN2B and PTPRD genes. JAK2, CD274, and PDCD1LG2 deletion often but not always coincided with CDKN2A/CDKN2B and PTPRD deletions. IFN-γ induced IRF1 expression and cell surface HLA-ABC levels in A549 cells that contain wildtype JAK2 but not in H1573 cells that had a truncating JAK2. Deletion of JAK2 or inhibition of JAK2 kinase activity in A549 cells resulted in loss of IFN-γ-regulated IRF1 and cell surface HLA-ABC, whereas expression of exogenous JAK2 in H1573 cells restored IFN-γ responses. These findings reveal a previously unrecognized significance of chromosome 9p deletion in NSCLC and point to chromosome 9p24.1 as a determinant of immune checkpoint therapy. Citation Format: Tao Shen, Jie Wu. Chromosome 9p24.1 deletions as a determinant of tumor immune surveillance and immune checkpoint blockade therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 585. doi:10.1158/1538-7445.AM2017-585

Genetic defects of the IRF1-mediated major histocompatibility complex class I antigen presentation pathway occur prevalently in the JAK2 gene in non-small cell lung cancer.

Recognition of major histocompatibility complex (MHC) class I antigens on tumor cells by cytotoxic T cells is involved in T cell-mediated tumor immune surveillance and immune checkpoint therapy. The interferon-γ (IFNγ)-IRF1 signaling pathway regulates MHC class I antigen presentation. To examine genetic defects of the IFNγ-IRF1 pathway in non-small cell lung cancer (NSCLC), we analyzed The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LuAd) and lung squamous cell carcinoma (LuSc) data. Loss-of-function (LOF) genetic alterations of the IFNγ-IRF1 pathway genes (IFNGR1, IFNGR2, JAK1, JAK2, STAT1, IRF1) were found in 64 (6.3%) of 1,016 patients. These genetic defects occur prevalently in JAK2 (33 cases) and often through deletions (29 cases) of chromosome 9p24.1. JAK2 deletions were frequently, but not always, associated with deletions of PD-L1 gene (CD274), PD-L2 gene (PDCD1LG2), PTPRD, and CDKN2A/CDKN2B at the chromosome 9p24.1-9p21.3 region. IRF1 expression was correlated with immune cytolytic activity markers GZMA and PRF1 in NSCLC. IFNγ induced IRF1 expression and cell surface HLA-A/HLA-B/HLA-C (HLA-ABC) in A549, H661, H292, and H2172 cells that contained the wildtype JAK2, but not in H1573 and H1623 cells that were JAK2 defective. Deletion of JAK2 or inhibition of the JAK2 kinase activity resulted in loss of IFNγ-induced IRF1 and cell surface HLA-ABC in JAK2 wildtype NSCLC cells, whereas expression of exogenous JAK2 in H1573 cells restored the IFNγ responses. These findings show that JAK2 deficiency is the major mechanism of genetic defects of the IFNγ-IRF1 pathway in NSCLC and reveal a previously unrecognized significance of chromosome 9p deletion in NSCLC.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents

AbstractPatients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Janus Kinase 2 (JAK2) Dissociates Hepatosteatosis from Hepatocellular Carcinoma in Mice

Hepatocellular carcinoma is an end-stage complication of non-alcoholic fatty liver disease (NAFLD). Inflammation plays a critical role in the progression of non-alcoholic fatty liver disease and the development of hepatocellular carcinoma. However, whether steatosis per se promotes liver cancer, and the molecular mechanisms that control the progression in this disease spectrum remain largely elusive. The Janus kinase signal transducers and activators of transcription (JAK-STAT) pathway mediates signal transduction by numerous cytokines that regulate inflammation and may contribute to hepatocarcinogenesis. Mice with hepatocyte-specific deletion of JAK2 (L-JAK2 KO) develop extensive fatty liver spontaneously. We show here that this simple steatosis was insufficient to drive carcinogenesis. In fact, L-JAK2 KO mice were markedly protected from chemically induced tumor formation. Using the methionine choline-deficient dietary model to induce steatohepatitis, we found that steatohepatitis development was completely arrested in L-JAK2 KO mice despite the presence of steatosis, suggesting that JAK2 is the critical factor required for inflammatory progression in the liver. In line with this, L-JAK2 KO mice exhibited attenuated inflammation after chemical carcinogen challenge. This was associated with increased hepatocyte apoptosis without elevated compensatory proliferation, thus thwarting expansion of transformed hepatocytes. Taken together, our findings identify an indispensable role of JAK2 in hepatocarcinogenesis through regulating critical inflammatory pathways. Targeting the JAK-STAT pathway may provide a novel therapeutic option for the treatment of hepatocellular carcinoma.

Adipocyte JAK2 mediates growth hormone-induced hepatic insulin resistance.

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue-dependent mechanism.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.