The JAK pathway is central to mammalian cell communication, characterized by rapid responses, receptor versatility, and fine-tuned regulation. It involves Janus kinases (JAK1, JAK2, JAK3, TYK2), which are activated when natural ligands bind to receptors, leading to autophosphorylation and activation of STAT transcription factors [1, 2]. JAK-dependent signaling plays a pivotal role in coordinating cell communication networks across a broad spectrum of biological systems including development, immune responses, cell growth, and differentiation. JAKs are frequently mutated in the aging hematopoietic system [3, 4] and in hematopoietic cancers [5]. Thus, dysregulation of the pathway results in various diseases, including cancers and immune disorders. The binding of extracellular ligands to class I and II cytokine receptors initiates a critical signaling cascade through the activation of Janus kinases (JAKs). Upon ligand engagement, JAKs become activated and phosphorylate specific tyrosine residues on the receptor, creating docking sites for signal transducer and activator of transcription (STAT) proteins. Subsequent JAK-mediated phosphorylation of STATs enables their dimerization and nuclear translocation, where they function as transcription factors to modulate gene expression. Under physiological conditions, JAK-signaling is a tightly regulated mechanism that governs cellular responses to external cues, such as cytokines and growth factors, ensuring homeostasis and maintaining the functional integrity of tissues and organs. Highly defined regulation of JAK-signaling is essential for balancing cellular responses to inflammatory stimuli and growth signals, thus safeguarding tissue health. In contrast, dysregulated JAK-signaling results in chronic inflammation and unrestrained cellular proliferation associated with various diseases. Understanding the qualitative and quantitative differences at the interface of physiologic JAK-signaling and its aberrant activation in disease is crucial for the development of targeted therapies that precisely tune this pathway to target pathologic activation patterns while leaving homeostatic processes largely unaffected. Consequently, pharmaceutical research has targeted this pathway for drug development leading to the approval of several substances with different selectivity profiles towards individual JAKs. Yet, the precise impact of inhibitor selectivity and the complex interplay of different functional modules within normal and malignant cells remains incompletely understood. In this review, we summarize the current knowledge on JAK-signaling in health and disease and highlight recent advances and future directions in the field.

The lysine-specific demethylase 1 (LSD1) regulates hematopoietic stem cell differentiation and has been identified as a therapeutic target in hematological disorders. LSD1 demethylates mono and dimethylated histones 3 at lysine 4 and 9. In addition, it acts as a scaffold for the formation of chromatin-modifying complexes that regulates the transcription of myeloid-lineage-specific genes in complex with GFI1, a transcriptional repressor. While both enzymatic and non-enzymatic functions of LSD1 have been well defined, the relative importance of these two functions in hematopoiesis remains incompletely understood. Here, we investigated the contribution of enzymatic and non-enzymatic functions of LSD1 to myelopoiesis. We show that myeloid differentiation is independent of the enzymatic functions of LSD1 but requires the non-enzymatic, scaffolding function, which directs GFI1 binding to target sequences. In the absence of the LSD1 protein, GFI1 DNA binding is diminished, and myeloid cell differentiation arrests at an immature, myelomonocytic-like cell stage, which overexpresses Prtn3. We provide functional data implicating Prtn3 as an effector of the stem cell expansion and myeloid maturation block caused by the loss of LSD1.

While pancreatic ductal adenocarcinoma (PDAC) carries a poor prognosis, a small fraction of patients show high microsatellite instability (MSI-H) and may respond to immune checkpoint inhibition. An MSI-H genotype is usually associated with deficiencies in the DNA mismatch-repair mechanism (MMRd). However, discordances between the mismatch-repair status by immunohistochemistry and the microsatellite status by molecular analyses have been noted. To date it is not clear whether PDAC patients with mutations in mismatch repair genes, which result in loss of protein expression (MMRd), who nonetheless retain microsatellite stability (MSS), can profit from checkpoint inhibitor therapy. Here, we present the case of a PDAC patient, diagnosed as MMRd/MSS, who responded to checkpoint inhibitor therapy after failing two lines of chemotherapy. Our data suggest that both MMR and microsatellite status should be determined in PDAC patients and that MMRd status alone, even in an MSS phenotype, can constitute an indication for checkpoint inhibitor therapy.

JAKoMo was a long-term, multicenter, non-interventional study observing the efficacy, safety, and quality of life (QOL) effects of ruxolitinib (RUX), managed per clinical routine at investigator discretion, for treatment of 943 patients with myelofibrosis (MF) in 122 German centers. Patients ≥ 18 years with a diagnosis of PMF or PPV-MF or PET-MF, who were suitable for in-label treatment with RUX were eligible and could be included either before (479 previously RUX-naïve [Arm A]) or after the start of treatment (464 RUX-experienced patients [Arm B]) and were followed over 36 months. Arm A showed rapid (≤ 6 months), sustained improvements from baseline in all efficacy outcomes and most QOL measures. Both arms showed an ~17% increase in the proportion of patients experiencing a normal German QOL during follow-up. Arm B entered the study with better outcomes and QOL than Arm A, with outcomes generally remaining stable over time. Adverse events were less common than in registrational trials, possibly due, in part, to lower real-world RUX dosing. Survival was comparable to published data. The JAKoMo study demonstrates that real-world RUX treatment of MF promotes significant and sustained clinical and QOL benefits, including improvement of general health and alleviation of MF-associated fatigue. Maximum sustained responses were generally achieved within 6 months and associated with fewer adverse events than in published randomized trials, which may reflect more conservative and personalized real-world dosing. Trial Registration: The JAKOMO trial: http://clinicaltrials.gov/show/NCT05044026.

Introduction CNL is a rare myeloproliferative neoplasm (MPN) with a 5-year-survival-rate of 28 percent, and no standard of care for treatment. The majority of CNL patients harbors the membrane proximal T618I mutation in the colony-stimulating factor 3 receptor (CSF3R), which leads to constitutive activation of the JAK1/STAT3-pathway. However, JAK1/2-inhibitor Ruxolitinib achieves only a limited response in CNL patients, suggesting the involvement of additional signaling pathways. Our previous data established that CSF3RT618I is retained in the endoplasmic reticulum (ER), where it interacts with Calnexin, induces unfolded protein response (UPR) and ER-Phagy. In this study we analyzed the role of ER-stress sensor ATF6 for CSF3Rmutations mediated cell proliferation and signaling, and explored potential signaling pathways crucial for Ruxolitinib-persistent CSF3RT618I expressing cells. Methods PDI inhibitor 16F16 treatments in Ba/F3, 32D and HEK293 cells were conducted to target ATF6 and analyze effects on membrane proximal-, truncation-, and compound-mutated CSF3R, and mutant Calreticulin (CALR)del52 mediated oncogenic signaling pathways, using MTT-assays, Western Blots (WB) and co-Immunoprecipitation (IP). RNA-seq was performed with Ba/F3 cells and peripheral blood samples from CNL-patients from the German Study Group for Myeloproliferative Neoplasms (GSG-MPN) (n=28). NIH-3T3 and 32D cells expressing CSF3RT618I, CSF3RWT-KDEL and CSF3R compound mutations were used to assess the significance of altered protein structure on signaling and UPR, using confocal microscopy, MTT-assays and WB. A PamGene assay was utilized to analyze differential kinase activity in Ruxolitinib-persistent CSF3RT618I expressing Ba/F3 cells. Results RNA-seq revealed upregulation of ER-stress-sensor ATF6 and ATF6 target genes in CSF3RT618I, CSF3RW791* and CSF3RT618I+Y748* expressing Ba/F3 cells, and in CNL-patients harboring CSF3RT618I or CSF3R-compound-mutations. Compound-mutated CSF3R proteins displayed a specific pattern of UPR induction compared to CSF3RT618I, suggesting an important role of the cytoplasmic tail of CSF3R in ER-stress-sensor activation. Mechanistically, PDI inhibitor 16F16 led to decreased ATF6 activation in 32D and Ba/F3 cells, and impaired dimerization of CSF3RT618I and CSF3RW783*, but not CSF3RWT expressing, flag-/HA-tagged CSF3R constructs containing, HEK293 cells. Targeting ATF6 activation and PDI activity with 16F16 led to a decrease in cell proliferation and reduced STAT3, Src kinase and AKT activation in membrane proximal-, truncation-, and compound-mutated CSF3R, as well as Ruxolitinib-persistent CSF3RT618I, but not CSF3RWTexpressing 32D and Ba/F3 cells. PamGene assay analysis revealed enhanced activity of PKC-family-kinases in Ruxolitinib-persistent CSF3RT618I expressing Ba/F3 cells compared to non-persisters. Co-IP showed interaction of CSF3RT618I and PKC-family-kinases and WB-results corroborated a JAK-independent activation mechanism. Interestingly, 32D cells expressing thrombopoietin receptor (MPL) and CALRdel52 also showed ATF6 protein activation and UPR induction. Treatment with 16F16 led to significantly decreased cell proliferation compared to CALRWT expressing 32D cells. ConclusionsCSF3RT618I strongly induces UPR through the PDI-ATF6-axis. Activation of this pathway requires specific regions of the cytoplasmic domain of CSF3R, acting in concert with protein folding alterations induced by T618I. Our results suggest a shift to enhanced PKC-family-kinase mediated signaling as a possible mechanism for CSF3RT618I expressing cells to evade Ruxolitinib mediated JAK1/2 inhibition, potentially explaining limited treatment efficacy in CNL patients. However, 16F16 treatment significantly decreases cell proliferation mediated by various CSF3R mutants, including Ruxolitinib-persistent CSF3RT618I expressing cells, potentially through a multilayered mechanism, comprising impaired dimerization and enhanced degradation of CSF3R mutants due to blockage of the PDI-ATF6-axis. CALRdel52 mediated proliferation, as seen in MPNs such as essential thrombocythemia, can also be diminished by 16F16. These results implicate the PDI-ATF6-axis as a novel and effective treatment target for CSF3R mutations mediated CNL and other MPNs.

Myeloproliferative neoplasms (MPN) harboring concurrent mutations in more than one driver gene represent an increasingly recognized yet frequently overlooked subgroup, posing significant diagnostic and clinical challenges. Current guidelines recommend a stepwise diagnostic approach: testing of CALR and MPL only if JAK2 is negative. This sequential strategy inherently risks overlooking cases, as simultaneous mutations in JAK2, CALR, and MPL, although rare, do exist. In published, rather limited datasets, patients typically exhibited older age and elevated platelet counts compared to those with single mutations, with suboptimal responses to conventional therapies necessitating tailored strategies. The true incidence of “double-driver” mutations is likely underestimated. To characterize the molecular landscape and clinical management of “double driver” patients 3 international patient cohorts were investigated: first we analyzed 84,921 samples with suspected diagnosis of MPN. 29,372 individuals (34.59%) were positive for at least one MPN driver mutation (JAK2, MPL or CALR). 439/29,372 individuals had at least 2 (1.5%) and 3/29,372 individuals all 3 driver mutations (0.01%) detectable. In 424 evaluable individuals, M:F ratio was 1.09 with a median age of 73.2 years (range 16.9-96). Leukocyte range was 1.5-117.0 Gpt/l and hemoglobin 6.1-22.0 g/dl, indicating a high variability depending on the clinical phenotype. Patients harboring 2 mutations frequently had elevated platelet counts (median 773Gpt/l; range 22-1,956 Gpt/l). Clone size of the respective driver mutations showed a high variability (CALR: mean 22.2%; range 0.85-62%; JAK2 16.9%, range 0.83-96%; MPL 13.5%, range 0.96-88.8%), while >80% of individuals revealed clone sizes <50%. 57% of cases that had received a complete NGS myeloid panel showed additional co-mutations, most frequently in TET2, SRSF2 and ASXL1, while 43% of the analyzed cases had no further mutation detectable. These results were confirmed in a cohort of the German MPN Study Group Registry, an observational study of MPN patients with >70 participating centers (university & community hospitals and office-based hematologists): out of 4,148 MPN patients with detailed clinical annotation, 54/4,148 (1.28%) were identified with 2 and 6/4,148 (0.05%) with all 3 driver mutations. Analysis of individually selected patient cells (with 2 confirmed drivers) in colony formation assays revealed their bi-clonal nature. The type of driver mutation combination had impact on the clinical phenotype with JAK2/MPL being predominantly diagnosed as ET (45%) and MF (40%), JAK2/CALR-co-mutated cases as ET (53%) and MF (22%), CALR/MPL-mutated cases as ET (60%) and MF (40%) and triple mutated cases as PV (50%) or ET (50%). Overall, patients with 2 drivers showed a higher rate of progression (to myelofibrosis or MPN-blast phase) compared to those with 1 driver mutation (16.7% vs. 9.9%). Of note, JAK2/CALR co-mutated PMF patients showed better overall survival (median OS: JAK2/CALR not reached; JAK2: 5.4 years, CALR: not reached), while JAK2/MPL co-mutation resulted in dismal survival (median OS: JAK2/MPL: 3.8 years; JAK2 5.4 years; MPL 3.2 years). Consistently, clonal competition assays conducted with murine hematopoietic cells over 8 days revealed clonal dominance of JAK2 over CALR (day 8; p<0.0046**) while MPL-mutated cells were dominant over JAK2-clones (day 8; p=0.0115*). Transcriptome profiling of competing cell populations identified distinct inflammatory signatures depending on the driver combination that can be traced to subclonal compartments in murine competition assays in vivo and single cell sequencing. To assess for therapy responses and clinical characteristics in more detail, we investigated 56 “double driver” patients with detailed clinical annotation from 12 international MPN centers and found differential clinical and molecular responses in patients treated with Jak-inhibitor or interferon-alpha therapies, e.g. partial and complete molecular responses of MPL-clones upon IFN-containing regimens. Our data provides insight into a rare, so far overlooked patient population and indicates a need to modify the sequential diagnostic approach. Appearance of MPL and CALR mutations parallel to JAK2 impact on prognosis irrespective of clone size. Differential patterns of therapy response (e.g. to IFN) appear to be relevant for molecular responses and survival.

Abstract Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA sequencing revealed that CALRdel52 led to the expansion of TGFβ1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGFβ antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, whereas T-cell depletion abrogated the protective effects conferred by neutralizing TGFβ. TGFβ1 reduced perforin and TNFα production by T cells in vitro. TGFβ1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGFβ1 expression. In four independent patient cohorts, TGFβ1 expression was increased in the BM of patients with MPN compared with healthy individuals, and the BM of patients with MPN contained a higher frequency of Treg compared with healthy individuals. Together, this study identified an ERK/Sp1/TGFβ1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell–mediated cytotoxicity. Significance: Targeting the mutant calreticulin/TGFβ1 axis increases T-cell activity and glycolytic capacity, providing the rationale for conducting clinical trials on TGFβ antagonists as an immunotherapeutic strategy in patients with myeloproliferative neoplasms.

Leukemia relapse is a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). We tested the potential of targeting T cell (Tc) immunoglobulin and mucin-containing molecule 3 (TIM-3) for improving graft-versus-leukemia (GVL) effects. We observed differential expression of TIM-3 ligands when hematopoietic stem cells overexpressed certain oncogenic-driver mutations. Anti-TIM-3 Ab treatment improved survival of mice bearing leukemia with oncogene-induced TIM-3 ligand expression. Conversely, leukemia cells with low ligand expression were anti-TIM-3 treatment resistant. In vitro, TIM-3 blockade or genetic deletion in CD8+ Tc enhanced Tc activation, proliferation, and IFN-γ production while enhancing GVL effects, preventing Tc exhaustion, and improving Tc cytotoxicity and glycolysis in vivo. Conversely, TIM-3 deletion in myeloid cells did not affect allogeneic Tc proliferation and activation in vitro, suggesting that anti-TIM-3 treatment-mediated GVL effects are Tc induced. In contrast to anti-programmed cell death protein 1 (anti-PD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) treatment, anti-TIM-3-treatment did not enhance acute graft-versus-host disease (aGVHD). TIM-3 and its ligands were frequently expressed in acute myeloid leukemia (AML) cells of patients with post-allo-HCT relapse. We decipher the connections between oncogenic mutations found in AML and TIM-3 ligand expression and identify anti-TIM-3 treatment as a strategy for enhancing GVL effects via metabolic and transcriptional Tc reprogramming without exacerbation of aGVHD. Our findings support clinical testing of anti-TIM-3 Ab in patients with AML relapse after allo-HCT.

Ruxolitinib (RUX) is a Janus kinase 1/2 inhibitor (JAKi) approved in the EU for treating disease‑related splenomegaly or symptoms in adults patients with myelofibrosis (MF). This is an interim analysis of JAKoMo, a prospective, non‑interventional, phase IV study in MF. Between 2012–2019 (cutoff March 2021), 928 patients (JAKi-naïve and -pretreated) enrolled from 122 German centers. This analysis focuses on JAKi-naïve patients. RUX was administered according to the Summary of Product Characteristics. Compared to the COMFORT-I, -II, and JUMP trials, patients in JAKoMo were older (median 73 years), had poorer Eastern Cooperative Oncology Group (ECOG) performance statuses (16.5

Leukemia stem cells (LSCs) share numerous features with healthy hematopoietic stem cells (HSCs). G-protein coupled receptor family C group 5 member C (GPRC5C) is a regulator of HSC dormancy. However, GPRC5C functionality in acute myeloid leukemia (AML) is yet to be determined. Within patient AML cohorts, high GPRC5C levels correlated with poorer survival. Ectopic Gprc5c expression increased AML aggression through the activation of NF-κB, which resulted in an altered metabolic state with increased levels of intracellular branched-chain amino acids (BCAAs). This onco-metabolic profile was reversed upon loss of Gprc5c, which also abrogated the leukemia-initiating potential. Targeting the BCAA transporter SLC7A5 with JPH203 inhibited oxidative phosphorylation and elicited strong antileukemia effects, specifically in mouse and patient AML samples while sparing healthy bone marrow cells. This antileukemia effect was strengthened in the presence of venetoclax and azacitidine. Our results indicate that the GPRC5C-NF-κB-SLC7A5-BCAAs axis is a therapeutic target that can compromise leukemia stem cell function in AML.

The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings.

Gastro-intestinal stromal tumors and acute myeloid leukemia induced by activating stem cell factor receptor tyrosine kinase (KIT) mutations are highly malignant. Less clear is the role of KIT mutations in the context of breast cancer. Treatment success of KIT-induced cancers is still unsatisfactory because of primary or secondary resistance to therapy. Mouse models offer essential platforms for studies on molecular disease mechanisms in basic cancer research. In the course of the Munich N-ethyl-N-nitrosourea (ENU) mutagenesis program a mouse line with inherited polycythemia was established. It carries a base-pair exchange in the Kit gene leading to an amino acid exchange at position 824 in the activation loop of KIT. This KIT variant corresponds to the N822K mutation found in human cancers, which is associated with imatinib-resistance. C3H KitN824K/WT mice develop hyperplasia of interstitial cells of Cajal and retention of ingesta in the cecum. In contrast to previous Kit-mutant models, we observe a benign course of gastrointestinal pathology associated with prolonged survival. Female mutants develop mammary carcinomas at late onset and subsequent lung metastasis. The disease model complements existing oncology research platforms. It allows for addressing the role of KIT mutations in breast cancer and identifying genetic and environmental modifiers of disease progression.

Even after development of the JAK1/JAK2 inhibitor ruxolitinib, myeloproliferative neoplasm (MPN) patients require novel therapeutic options. While ruxolitinib can considerably improve quality of life and prolong survival, it does not modify the natural disease course in most patients. Moreover, resistance develops with prolonged use. Therefore, various combination treatments are currently being investigated. Published data provide a compelling rationale for the inhibition of insulin growth factor-1 receptor (IGF-1R) signaling in MPN. Here we report that genetic and pharmacological inhibition of IGF-1R selectively reduced Jak2(V617F)-driven cytokine-independent proliferation ex vivo. Two different structurally unrelated IGF-1R inhibitors ameliorated disease phenotype in a murine MPN model and significantly prolonged survival. Moreover, in mice, low-dose ruxolitinib synergized with IGF-1R inhibition to increase survival. Our data demonstrate preclinical efficacy of IGF-1R inhibition in a murine MPN model.

Inflammation-induced thrombosis represents a severe complication in patients with myeloproliferative neoplasms (MPN) and in those with kidney dysfunction. Overlapping disease-specific attributes suggest common mechanisms involved in MPN pathogenesis, kidney dysfunction, and thrombosis. Data from 1420 patients with essential thrombocythemia (ET, 33.7%), polycythemia vera (PV, 38.5%), and myelofibrosis (MF, 27.9%) were extracted from the bioregistry of the German Study Group for MPN. The total cohort was subdivided according to the calculated estimated glomerular filtration rate (eGFR, (mL/min/1.73 m2)) into eGFR1 (≥90, 21%), eGFR2 (60–89, 56%), and eGFR3 (<60, 22%). A total of 29% of the patients had a history of thrombosis. A higher rate of thrombosis and longer MPN duration was observed in eGFR3 than in eGFR2 and eGFR1. Kidney dysfunction occurred earlier in ET than in PV or MF. Multiple logistic regression analysis identified arterial hypertension, MPN treatment, increased uric acid, and lactate dehydrogenase levels as risk factors for kidney dysfunction in MPN patients. Risk factors for thrombosis included arterial hypertension, non-excessive platelet counts, and antithrombotic therapy. The risk factors for kidney dysfunction and thrombosis varied between MPN subtypes. Physicians should be aware of the increased risk for kidney disease in MPN patients, which warrants closer monitoring and, possibly, early thromboprophylaxis.

The vast majority of patients with myeloproliferative neoplasms (MPN), polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis acquire driver mutations in the JAK2, MPL or CALR gene. Clustering of MPN is seen in select families, but in most pedigrees the MPN-predisposing change has not been determined and affected individuals somatically acquire one of the three above-mentioned driver mutations. In contrast, a small number of individuals with hereditary thrombocythemia (HT) carry constitutive alterations, e.g., in the TPO or the LNK (SH2B3) gene. Acquired mutations in LNK, a negative regulator of JAK2 signaling, rarely occur in both sporadic and familial MPN cases. In the latter, they do not segregate with disease phenotype and diseased individuals acquire a concomitant MPN driver mutation. It, therefore, appears unlikely that mutant LNK acts as a driver in MPN. We have shown that the transcription factor Nuclear Factor Erythroid-derived-2 (NFE2) is overexpressed in the vast majority of MPN patients, independent of other molecular aberrations. In addition, we have identified NFE2 mutations in MPN and acute myeloid leukemia (AML) patients, that enhance the activity of wild-type (WT) NFE2. In several murine models, elevated NFE2 activity causes pathognomonic features of MPN. The molecular mechanisms by which NFE2 mutants exert their effect remain unclear since the regulation of NFE2 transcriptional activity is poorly understood. Various post-translational modifications have been described, including ubiquitination, phosphorylation, and sumoylation but their functional role remains elusive. Here, we identified a 74-year-old female patient given the diagnosis of ET by World Health Organization criteria (Online Supplementary Table S1), who tested negative for the three MPN driver mutations, JAK2, CALR, and MPL. Sequencing 36 myeloid neoplasms associated genes (Online Supplementary Tables S2 and S3) revealed both a previously described p.E208Q point-mutation in LNK as well as a novel mutation in NFE2 (c.1102A>T) that prematurely truncates the protein at lysine 368 (p.K368X, Figure 1A). Buccal swab DNA analysis determined that both mutations were heterozygously present in the germline. Because of the constitutive nature of both mutations, this patient should be designated as having hereditary thrombocythemia. The p.K368X mutation leaves almost the entire NFE2 protein, including the bZIP domain and the N-terminal activation domain, intact. Only the terminal 4 amino acids are lost. Notably, this mutation deletes the ψKXE sumoylation consensus motif identified at lysine 368 and shown to be sumoylated by SUMO1 in vitro and in vivo. The LNK p.E208Q mutation retains near-complete inhibitory capacity and did not confer significantly higher TPO-hypersensitivity in cell proliferation assays than WT-LNK, suggesting only a subtle loss of function. Therefore, we hypothesized that the loss of sumoylation increases NFE2 activity, which, in co-operation with mutant LNK, drives thrombocytosis in this patient. To test whether NFE2-K368X retains binding to its cognate DNA motif, we performed an electromobility shift assay (EMSA) using a consensus NFE2 binding site from the human PBGD promoter. DNA binding is preserved in the NFE2-K368X mutant (Figure 1B), consistent with retention of the complete DNA binding and heterodimerization domains. We subsequently examined the ability of NFE2-K368X to transactivate transcription using a luciferase reporter assay. Heterodimerization with MafG is required for optimal NFE2 activity (Online Supplementary Figure S1). The NFE2-K368X mutant was more than twice as active at promoting reporter gene expression than WT-NFE2 (Figure 1C). Because transcription off a plasmid DNA template does not model intact chromatin, we used endogenous gene activation in a cell line as a second read-out. CB3 cells are devoid of NFE2 expression due to viral integration but express β-globin upon re-introduction of NFE2. We therefore lentivirally transduced CB3 cells with either WT-NFE2 or NFE2-K368X and determined β-globin expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Again, NFE2-K368X, present in the same amount, was two times more active than its WT counterpart in directing transcription, demonstrating that the mutation results in a protein with supraphysiological activity on intact chromatin (Figure 1D). NFE2-K368X thus constitutes a novel Type Ia mutation, DNA–binding and activating, according to the classification of NFE2 mutations we proposed. To investigate sumoylation of the NFE2-K368X mutant, we conducted in vitro sumoylation assays using recombinantly expressed proteins. SUMO is attached to proteins by hierarchical action of the E1-activating enzyme Aos1/Uba2, the E2-conjugating enzyme UBC9, and a substrate-specific E3-ligase. Sumoylation of WTNFE2 with SUMO1 has been shown in assays that contained Aos1/Uba2 and UBC9 but lacked an E3-ligase. Under these conditions, we could not detect SUMO1 modification of NFE2 (Online Supplementary Figure S2). Substrate recognition is accomplished by UBC9, but E1 and E2 enzymes have poor transfer efficiency, which is stimulated by E3-ligases. Addition of the E3-ligases IR1+M, PIAS1, or ZNF451-N, led to sumoylation of GST-NFE2-WT but not the NFE2-K368X mutant (Figure 2, top). IR1+M, the catalytic core domain of the E3-enzyme RanBP2, has high SUMO ligase activity but low substrate specificity. Unphysiological in vitro conditions can facilitate sumoylation of non-canonical lysine residues, which may facilitate both the observed IR1+M mediated sumoylation of WT-NFE2 (Figure 2, lane 1, marked*) as well as unspecific modification of the NFE2-K368X mutant (Figure 2, lane 6, marked**). The RanBP2 fragment RanBP2DFG has a higher substrate specificity, and did not sumoylate either GST-NFE2-WT or NFE2-K368X (Figure 2, lanes 2 and 7). These data suggest that while NFE2 sumoylation is facilitated by the minimal catalytic activity of the IR1+M fragment, NFE2 is not a substrate of the RanBP2 E3-ligase itself. Presence of the small subunit MafG does not influence the sumoylation efficacy of NFE2 by IR1+M with SUMO1 (Online Supplementary Figure S3). Two additional E3-ligases modified NFE2: PIAS1 and ZNF451-N, both with either SUMO1 or SUMO2/3 (Figure 2, top and bottom, lanes 3 and 4). The ZNF451-N ligase has been described as specific for SUMO2/3, but modified GST-NFE2-WT with SUMO1 in our study. This activity may result from the high concentration of the components and the unphysiological conditions in vitro. PIAS1 is a member of the PIAS family class of E3-ligases and was described to facilitate MafG sumoylation by

Histone methylation tightly regulates chromatin accessibility, transcription, proliferation, and cell differentiation, and its perturbation contributes to oncogenic reprogramming of cells. In particular, many myeloid malignancies show evidence of epigenetic dysregulation. Jumonji C (JmjC) domain-containing proteins comprise a large and diverse group of histone demethylases (KDMs), which remove methyl groups from lysines in histone tails and other proteins. Cumulating evidence suggests an emerging role for these demethylases in myeloid malignancies, rendering them attractive targets for drug interventions. In this review, we summarize the known functions of Jumonji C (JmjC) domain-containing proteins in myeloid malignancies. We highlight challenges in understanding the context-dependent mechanisms of these proteins and explore potential future pharmacological targeting.

A central challenge in the care of patients with myeloproliferative neoplasms (MPNs) is identifying those individuals at high risk for transformation into acute leukemia, an exacerbation that carries a very poor prognosis.(1) In this issue of Blood, Marcault and colleagues(2) describe a novel type of mutation, for which I propose the term "sentinel mutation" (see figure). The acquisition of a "sentinel mutation" drastically increases the likelihood of leukemic transformation, even though in some patients the mutation does not occur in the cells that form the leukemic clone. "Sentinel mutations" function like roses planted in vineyards. Ailing flowers forecast the vines' impending infection with black rot or downy mildew as the roses are affected first, even if the species that blight the flowers differ slightly from those that spoil the grapes. Similarly, Marcault and colleagues have shown that patients with MPN that acquire mutations in the transcription factor "nuclear factor erythroid 2" (NFE2) carry an increased risk of leukemic transformation, even though in some patients the mutations are not found in the leukemic cells.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myelo-proliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders. (C) 2020 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Of BCR-ABL negative myeloproliferative neoplasm (MPN) patients, 3-14 % display a concomitant monoclonal gammopathy (MGUS). Nonetheless, literature on co-occurring MPN and MGUS is scarce, the molecular underpinnings are unknown and it is unclear whether patients require a specific management. Here, we compared the clinical and genetic features of MPN patients with and without concomitant MGUS. Of 114 MPN patients prospectively studied by serum immunofixation (median age, 67 years; 36.0 % essential thrombocythemia [ET], 24.6 % polycythemia vera [PV], 11.4 % secondary myelofibrosis [sMF], 28.1 % primary myelofibrois [PMF]; 73.7 % JAK2 V617F positive), 10 (9 %) harbored an M-protein. No relevant clinical differences existed between MPN patients with or without M-protein. Seven additional MPN/MGUS patients were retrospectively identified in our MPN registry, yielding a total of 17 patients (7 ET, 3 PV, 3 sMF, 4 PMF). One patient developed multiple myeloma (MM) and one smoldering MM. Seven of 12 patients analyzed carried mutations (e.g. in ASXL1 or TET2) in addition to those in JAK2 or CALR, and 4 of 10 patients showed aberrant cytogenetics. M-protein was mainly IgG (12/17), followed by IgM (4/17). In the two patients that underwent allogeneic stem cell transplantation mutant JAK2 and M-protein were no longer detectable post-transplant. In conclusion, MGUS prevalence in our cohort was in the range of previous reports and at most slightly higher than expected in the general population. MGUS presence did not correlate with a specific MPN entity, clinical features or genetic alterations. Our observations suggest that there is no strong clinical or biological relationship between the occurrence of MGUS and MPN.

The histone demethylase JMJD1C is overexpressed in patients with myeloproliferative neoplasms (MPNs) and has been implicated in leukemic stem cell function of MLL-AF9 and HOXA9-driven leukemia. In the emerging field of histone demethylase inhibitors, JMJD1C therefore became a potential target. Depletion of Jmjd1c expression significantly reduced cytokine-independent growth in an MPN cell line, indicating a role for JMJD1C in MPN disease maintenance. Here, we investigated a potential role for the demethylase in MPN disease initiation. We introduced a Cre-inducible JAK2V617F mutation into Jmjd1c knockout mice. We show that Jmjd1c is dispensable, both for healthy hematopoiesis as well as for JAK2V617F-driven MPN disease initiation. Jmjd1c knockout mice did not show any significant changes in peripheral blood composition. Likewise, introduction of JAK2V617F into Jmjd1c-/- mice led to a similar MPN phenotype as JAK2V617F in a Jmjd1c wt background. This indicates that there is a difference between the role of JMJD1C in leukemic stem cells and in MPN. In the latter, JMJC domain-containing family members may serve redundant roles, compensating for the loss of individual proteins.