Calreticulin (CALR) mutations are prevalent in 20%-30% of patients with BCR::ABL1-negative myeloproliferative neoplasms (MPN). Mutant calreticulin (mutCALR), presented by the thrombopoietin receptor (MPL, also known as TPOR or CD110) on the surface of the disease-initiating MPN progenitors, represents an ideal target for curative immunotherapies including monoclonal antibodies, bispecific T cell engaging antibodies (TCE), and CAR-T cell therapies. Despite that two clinical TCE candidates have advanced into phase 1 trials in recent 2 years, depletion of mutCALR+ hematopoietic stem cells and normalization of hematopoiesis remained absent in preclinical evaluation. Here, we developed a bispecific T cell engager DX1-2C11 that specifically and efficiently eradicates mutCALR-expressing cells via recruiting polyclonal T cells. DX1-2C11 depleted Ba/F3 cells expressing mutCALR, as well as primary murine myeloid cells in a dose-dependent manner in vitro. In CALRdel52 transgenic mice, a single dose of DX1-2C11 activated CD4+ and CD8+ T cells in the peripheral blood, spleen and bone marrow within 24 h. Furthermore, a single dose of DX1-2C11 reduced platelet counts in the periphery and decreased mutant stem/progenitor cell populations in the spleen and bone marrow by Day 7 posttreatment. Notably, the reduction of mutant burden was durably maintained in secondary recipient mice. In the disseminated NSG model, DX1-2C11 delivered immediate tumor burden reduction and significantly prolonged the overall survival of mice compared to the control group. Taken together, these data suggest that bispecific T cell engaging antibody targeting mutCALR represents a curative strategy that efficiently eliminates mutant MPN stem cells in vivo.

Acute myeloid leukemia (AML) is a complex hematological malignancy with multiple disease sub-groups defined by somatic mutations and heterogeneous outcomes. Although genome-wide association studies (GWAS) have identified a small number of common genetic variants influencing AML risk, the heritable component of this disease outside of familial susceptibility remains largely undefined. Here we perform a meta-analysis of four published GWAS plus two new GWAS, totalling 4710 AML cases and 12938 controls. We identify a new genome-wide significant risk locus for pan-AML at 2p23.3 (rs4665765; P=1.35x10-8; EFR3B, POMC, DNMT3A, DNAJC27) which also significantly associates with patient survival (P=6.09x10-3). Our analysis also identifies three new genome-wide significant risk loci for disease sub-groups, including AML with deletions of chromosome 5 and/or 7 at 1q23.3 (rs12078864; P=7.0x10-10; DUSP23) and cytogenetically complex AML at 2q33.3 (rs12988876; P=3.28x10-8; PARD3B) and 2p21 (rs79918355; P=1.60x10-9; EPCAM). We also investigated loci previously associated with risk of clonal hematopoiesis (CH) or clonal hematopoiesis of indeterminate potential (CHIP) and identified several variants associated with risk of AML. Our results further inform on AML etiology and demonstrate the existence of disease sub-group specific risk loci.

During antiviral immune responses, activated immune cells remodel metabolic pathways towards uptake and utilization of biosynthetic and bioenergetic metabolites. Concurrently, viral infections alter metabolic environments, impacting metabolite availability for the establishment of an effective immune response. Here, we integrated in vivo metabolomics data from murine and human viral infections with in vitro metabolite screens, identifying purine nucleobases as novel immunometabolites that enhance CD8+ T cell effector function. We found that CD8+ T cells can switch from resource-intensive purine de novo synthesis to purine salvage pathway, to produce nucleotides from purine nucleobases. This strategy of metabolic adaptation allows diversion of biosynthetic and bioenergetic resources towards enhancing effector molecule production. Our findings unveil an adaptation strategy by CD8+ T cells to manage metabolic challenges in dynamic organismal environments and suggest pharmacological targets in purine metabolism as potential targets for immunotherapy. ![Figure][1] Graphical Abstract Instead of producing nucleotides via purine de novo synthesis, CD8+ T cells can import and utilize purine nucleobases via the purine salvage pathway to divert bioenergetic and biosynthetic resources towards effector function. By shifting from purine de novo synthesis to the purine salvage pathway, cells save significant resources: 5 moles of the key bioenergetic metabolite ATP, and biosynthetic metabolites including 2 moles of glutamine, 1 mole each of serine or glycine, and 1 mole of aspartate. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 677006 ANR PRC MetaNiche, ANR-22-CE15-0015-02 [1]: pending:yes

Genetically engineered mouse models have the potential to unravel fundamental biological processes and provide mechanistic insights into the pathogenesis of human diseases. We have previously observed that germline genetic variation at the TULP4 locus influences clinical characteristics in patients with myeloproliferative neoplasms. To elucidate the role of TULP4 in pathological and physiological processes in vivo, we generated a Tulp4 knockout mouse model. Systemic Tulp4 deficiency exerted a strong impact on embryonic development in both Tulp4 homozygous null (Tulp4-/-) and heterozygous (Tulp4+/-) knockout mice, the former exhibiting perinatal lethality. High-resolution episcopic microscopy (HREM) of day 14.5 embryos allowed for the identification of multiple developmental defects in Tulp4-/- mice, including severe heart defects. Moreover, in Tulp4+/- embryos HREM revealed abnormalities of several organ systems, which per se do not affect prenatal or postnatal survival. In adult Tulp4+/- mice, extensive examinations of hematopoietic and cardiovascular features, involving histopathological surveys of multiple tissues as well as blood counts and immunophenotyping, did not provide evidence for anomalies as observed in corresponding embryos. Finally, evaluating a potential obesity-related phenotype as reported for other TULP family members revealed a trend for increased body weight of Tulp4+/- mice.Research Highlights center dot To study the role of the TULP4 gene in vivo, we generated a Tulp4 knockout mouse model.center dot Correlative analyses involving HREM revealed a strong impact of Tulp4 deficiency on murine embryonic development.

BackgroundThe expansion of hematopoietic stem cells caused by acquired somatic mutations (clonal hematopoiesis [CH]) is a novel cardiovascular risk factor. The prognostic value of CH in patients with carotid atherosclerosis remains to be evaluated.ObjectivesThis study assessed the prognostic significance of CH in patients with atherosclerosis as detected by ultrasound of the carotid artery.MethodsWe applied deep sequencing of selected genomic regions within the genes DNMT3A, TET2, ASXL1, and JAK2 to screen for CH in 968 prospectively collected patients with asymptomatic carotid atherosclerosis evaluated by duplex sonography.ResultsWe detected clonal markers at variant allele frequency ≥2% in 133 (13.7%) of 968 patients (median age 69.2 years), with increasing prevalence at advanced age. Multivariate analyses including age and established cardiovascular risk factors revealed overall presence of CH to be significantly associated with increased risk of cardiovascular death (HR: 1.50; 95% CI: 1.12-2.00; P = 0.007), reflected also at the single gene level. The effect of CH was more pronounced in older patients and independent of the patients’ inflammatory status as measured by high-sensitivity C-reactive protein. Simultaneous assessment of CH and degree of carotid stenosis revealed combined effects on cardiovascular mortality, depicted by a superior risk for patients with >50% stenosis and concomitant CH (adjusted HR: 1.60; 95% CI: 1.08-2.38; P = 0.020).ConclusionsCH status in combination with the extent of carotid atherosclerosis jointly predict long-term mortality. Determination of CH can provide additional prognostic information in patients with asymptomatic carotid atherosclerosis.

Topic: 16. Myeloproliferative neoplasms - Clinical Background: According to recent real-world evidence, patients with high-risk polycythemia vera (PV) receiving standard care spend <30% of time in response for blood count targets recommended by European LeukemiaNet (ELN) (Carpenter et al 2022; eJHaem). Individual responses fluctuated considerably; a novel observation not detectable by conventional evaluation at predefined timepoints. Ropeginterferon alfa-2b induces higher response rates than standard treatment (hydroxyurea [HU]/best available treatment [BAT]) after long-term therapy, but whether this corresponds with cumulative time in response is unknown. Therefore, final PROUD-PV/CONTINUATION-PV data were evaluated regarding time in peripheral blood count remission. Aims: To assess the cumulative proportion of time in response for blood counts according to ELN targets in patients treated with ropeginterferon alfa-2b vs HU/ BAT over a period of ≥6 years. Methods: Patients with PV (WHO 2008 criteria) who were HU-naïve/pre-treated and gave informed consent were randomized 1:1 to ropeginterferon alpha-2b or control (HU) for one year in PROUD-PV (EudraCT: 2012-005259-18). Dosing was optimized per protocol in both arms. In the extension CONTINUATION-PV (2014-001357-17), patients in the control arm could switch from HU to BAT. Hematology was evaluated at intervals of 2-12 weeks for 5 years, and 6-monthly in the final year. Complete hematologic response (CHR) was defined per modified ELN criteria (hematocrit <45% with no phlebotomy for ≥3 months, PLT<400x109/L and WBC<10x109/L). Cumulative time in CHR and in response for individual parameters were assessed in the CONTINUATION-PV full analysis set based on the last response at each assessment, up to study completion/discontinuation. All safety data were analyzed. Results: The full analysis set comprised 169 patients: 95 in the ropeginterferon alfa-2b arm and 74 in the control arm. Individual patient data for time spent in CHR over ≥6 years demonstrate more consistent blood count responses among patients treated with ropeginterferon alfa-2b than in the standard treatment arm, in which 88% received HU as of the last assessment (Figure 1). Despite a more gradual onset of response in the ropeginterferon alfa-2b arm, on average, patients spent 60.9% of time in CHR vs. 41.2% for patients in the control arm (p=0.0437). These findings align with results of log-binomial modelling of longitudinal blood count assessments over the entire 6 years, indicating a higher CHR rate for ropeginterferon alfa-2b vs. standard treatment (RR: 1.27 [95% C.I. 1.05 to 1.53; p=0.0152]). Differences were also observed for individual parameters, with significantly more time spent with WBC counts on target in the ropeginterferon alfa-2b arm vs. control (mean: 93.7% vs. 80.9% p=0.0249). The respective mean proportion of time in response was 65.1% vs 57.0% (p=0.4501) for hematocrit and 87.7% vs 75.6% (p=0.0875) for PLT counts. Including PROUD-PV (N=254), 5 thromboembolic events occurred in the ropeginterferon alfa-2b arm; CHR was recorded at the preceding visit in only 1 case. On standard treatment, 7 thromboembolic events occurred, 3/7 preceded by CHR. Statistical correlation with CHR was precluded by the low incidence of events. Summary/Conclusion: These results underscore the difficulty of maintaining PV patients within peripheral blood count targets during standard treatment and indicate that more consistent control of these parameters can be achieved with ropeginterferon alfa-2b. Thromboembolic events were rare during ropeginterferon alfa-2b therapy and largely occurred during periods of non-response, in agreement with current response criteria.Keywords: Myeloproliferative disorder, Interferon alpha, Polycythemia vera

Myeloproliferative neoplasms (MPNs) are clonal malignant disorders of hematopoiesis arising in the hematopoietic stem cell (HSC) compartment that are characterized by excessive production of mature blood cells of the myeloid lineage. Transformation to secondary acute myeloid leukemia (sAML) represents a significant cause of death among MPN patients and this transformation occurs mainly from the clone carrying the disease phenotype driver mutation. Current treatment options for MPN patients are not curative and are limited to symptomatic treatment. Therefore, identification of novel therapeutic approaches with a clear disease-modifying effect for the treatment of MPNs and intercepting their progression to sAML is an unmet medical need. Mutations in JAK2, thrombopoietin receptor (MPL), and calreticulin (CALR) are phenotypic drivers in the pathogenesis of MPN. CALR mutations (CALRmut) are the second most frequent in MPN. CALRmut are insertions or deletions resulting in a frameshift in the last exon of the gene, causing a loss of the KDEL ER-retention motif and generation of a 36 amino acid positively charged C-terminal neoantigen. Due to loss of the KDEL motif, CALRmut are not confined to the ER and through interaction with MPL are trafficked to the cell surface where they induce persistent MPL activation and oncogenicity. Immunotherapies engaging T cells, such as bispecific cluster of differentiation 3 (CD3) redirection antibodies, show promising response rates in the clinic. Here, we describe JNJ-88549968, a novel first in class T-cell redirecting bispecific antibody that selectively targets CALRmut with the potential to achieve cures by eliminating MPN clones. The mechanism of action of JNJ-88549968 is to act as a bridge between CALRmut MPN cancer cells and T cells, inducing T-cell activation with subsequent T-cell-mediated cytotoxicity to CALRmut cancer cells in vitro and in vivo. JNJ-88549968 recognizes CALRmut epitopes common to all known CALRmut types. Cell surface localization of CALRmut was confirmed in CD34 + cells from CALRmut MPN patients. In-depth characterization of T cells from CALRmut MPN patients, using CyTOF and functional assays, confirmed their fitness and functionality. These data validate T-cell redirection as a rational therapeutic strategy for MPN patients carrying CALRmut. JNJ-88549968 demonstrated selective binding to CALRmut cell lines and no measurable binding to CALR wild type cells. JNJ-88549968 led to CALRmut-selective T-cell activation and cytotoxicity to CALRmut-engineered cell lines in vitro. The activity of JNJ-88549968 was also explored in an autologous setting using CD34 + cells isolated from CALRmut MPN patients as target cells together with T cells isolated from the same patients. JNJ-88549968 elicited concentration-dependent cytotoxicity of patient-derived CALRmut CD34 + cells. JNJ-88549968-mediated cytotoxicity was observed against all tested CALRmut CD34 + cancer cells and was independent of the type of CALR mutation. Moreover, JNJ-88549968 mediated robust in vivo efficacy in two independent CALRmut-positive xenograft murine leukemia models. In an established disseminated model, treatment with JNJ-88549968 significantly increased lifespan (ILS) compared to vehicle-treated control mice. Secreted CALRmut protein can be found in CALRmut MPN patient plasma. Evaluation of the impact of soluble CALRmut on JNJ-88549968 activity, including CALRmut-patient derived whole blood studies, indicated no effect on the activity of JNJ-88549968 in vitro. Taken together, JNJ-88549968 is a novel first-in-class bispecific T-cell redirection antibody investigated for the treatment of CALRmut MPN. JNJ-88549968 is currently being advanced for clinical investigation in patients with MPN.

Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.

BCR::ABL1-negative myeloproliferative neoplasms (MPNs) are clonal diseases originating from a single hematopoietic stem cell that cause excessive production of mature blood cells. The 3 subtypes, that is, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are diagnosed according to the World Health Organization (WHO) and international consensus classification (ICC) criteria. Acquired gain-of-function mutations in 1 of 3 disease driver genes (JAK2, CALR, and MPL) are the causative events that can alone initiate and promote MPN disease without requiring additional cooperating mutations. JAK2-p.V617F is present in >95% of PV patients, and also in about half of the patients with ET or PMF. ET and PMF are also caused by mutations in CALR or MPL. In ∼10% of MPN patients, those referred to as being "triple negative," none of the known driver gene mutations can be detected. The common theme between the 3 driver gene mutations and triple-negative MPN is that the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway is constitutively activated. We review the recent advances in our understanding of the early events after the acquisition of a driver gene mutation. The limiting factor that determines the frequency at which MPN disease develops with a long latency is not the acquisition of driver gene mutations, but rather the expansion of the clone. Factors that control the conversion from clonal hematopoiesis to MPN disease include inherited predisposition, presence of additional mutations, and inflammation. The full extent of knowledge of the mutational landscape in individual MPN patients is now increasingly being used to predict outcome and chose the optimal therapy.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 com-plex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Introduction: Ropeginterferon alfa-2b at a fixed dose of 100 µg/2 weeks was efficacious in patients with low-risk polycythemia vera (PV) in the Low-PV trial, but the response rate was lower in a group of patients who switched to ropeginterferon alfa-2b after failing to respond to phlebotomy alone (Barbui et al., 2023). The finding that this group had a higher proportion of males and higher baseline values for body-mass index, phlebotomy demand, blood counts and JAK2V617F allele burden suggests that these characteristics might predict a higher dose requirement of ropeginterferon alfa-2b. We analyzed low-risk PV patients from the PROUD-PV/CONTINUATION-PV (NCT01949805/NCT02218047) cohort to examine the impact of baseline characteristics and individually optimized dose levels of ropeginterferon alfa-2b on response status at 12, 24 and 72 months. Methods: Patients diagnosed with PV (WHO 2008 criteria) were randomized 1:1 and received ropeginterferon alfa-2b or standard care for a total of ≥6 years during the 12-month PROUD-PV study and its extension CONTINUATION-PV. Low-risk patients (aged ≤60 years with no history of thrombosis) allocated to the ropeginterferon alfa-2b arm were analyzed. Ropeginterferon alfa-2b was started at a dose of 100 µg/2 weeks (50 µg if transitioning from hydroxyurea) and escalated by 50 µg/2 weeks until blood counts normalized to a maximum of 500 µg/2 weeks. Grade 2-3 drug-related toxicity mandated dose reduction/interruption. During maintenance treatment the dosing interval could be extended to 3-4 weeks. Complete hematologic response (CHR) defined by modified ELN criteria (without the spleen criterion) and safety data at 12, 24 and 72 months were assessed. Results: In the setting of individualized dosing, 33/56 (58.9%) low-risk PV patients allocated to ropeginterferon alfa-2b in PROUD-PV achieved a CHR at month 12. Response rates increased with longer treatment duration; of the 46 low-risk patients enrolled in CONTINUATION-PV; 80.4% and 73.2% of patients with available data achieved a CHR at months 24 and 72, respectively. Baseline characteristics by response status at month 12 indicated that males were overrepresented among non-responders (15/23 non-responders [65.2%] were male vs 11/33 responders [33.3%] [p=0.0291]), and baseline median JAK2V617F allele burden was non-significantly higher in non-responders at 12 months (45.6% vs 35.5%: p=0.1860). However, no significant differences in these baseline parameters were observed by long-term response status (24 months or 72 months), nor were there consistent differences in baseline body mass index or blood counts according to response status at any time point evaluated. The most frequent reason for non-response was failure to meet the CHR criterion hematocrit <45% without phlebotomy in the past 3 months, accounting for the majority of non-responders at months 12, 24 and 72 (13/23 [56.5%], 6/9 [66.7%], and 6/11 [54.5%], respectively), followed by discontinuation for any reason, which accounted for 30.4% of non-responders at month 12 and 36.4% at month 72. Only one low-risk patient discontinued due to ropeginterferon alfa-2b-related toxicity. Despite response-driven dosing, the median 4-weekly derived dose of ropeginterferon alfa-2b in the first year was only somewhat higher in non-responders compared with responders (809 µg [range: 209-898] vs 717 µg [188-898]; p=0.6954); 10/23 of non-responders at month 12 did not escalate the dose immediately according to protocol on ≥1 occasion during the first year, e.g. due to responses already observed for leukocyte or platelet counts. In the second year, the median 4-weekly doses were 997 µg (range: 211-997) vs 690 µg (88-1036) respectively (p=0.2024), indicating that non-responders received and tolerated the maximum dose (500 µg/2 weeks). Conclusions: High long-term response rates (80.4% at 24 months; 73.2% at 72 months) were achieved with ropeginterferon alfa-2b treatment in the low-risk PV population when the dose was optimized on an individual basis. No specific baseline characteristics among low-risk PV patients appear to be associated with long-term response to ropeginterferon alfa-2b. However, the PROUD-PV/CONTINUATION-PV studies show that - as hypothesized based on the Low-PV trial data - some low-risk patients require and can tolerate high doses, and that the optimal dose of ropeginterferon alfa-2b varies substantially between patients.

Background: Treatment of polycythemia vera (PV) aims to prevent thromboembolic complications, reduce the risk of progression to acute leukemia or myelofibrosis - of particular concern to patients - and ameliorate the symptom burden; specifically, to improve quality of life, therapy should address the most clinically important symptoms while reducing phlebotomies to avoid iron-deficiency symptoms. Long-term efficacy and safety of ropeginterferon alfa-2b have been demonstrated in PROUD-PV/CONTINUATION-PV; the final analysis applied a patient-focused approach. Aims: To analyze the patient-relevant benefit of ropeginterferon alfa-2b versus hydroxyurea (HU)/best available treatment (BAT) over 6 years. Methods: Patients diagnosed with PV according to WHO 2008 criteria who were cytoreduction-naïve or hydroxyurea pre-treated and gave written informed consent were randomized 1:1 to ropeginterferon alpha-2b or control treatment (HU) for one year in PROUD-PV. In CONTINUATION-PV, control arm patients could switch from HU to BAT. Patient-reported PV symptom burden was assessed based on adverse events documented in the patient diary and recorded at each visit; items defined in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (MPN-SAF TSS; fatigue, concentration problems, early satiety, inactivity, night sweats, itching, bone pain, abdominal discomfort, weight loss, and fevers) and medical synonyms were evaluated post-hoc. Efficacy assessments included Kaplan-Meier analysis of event-free survival, phlebotomy need and JAK2V617F allele burden. Analyses were conducted on the CONTINUATION-PV full analysis set over 6 years of treatment. Results: The full analysis set comprised 95 patients in the ropeginterferon alfa-2b arm and 74 in the control arm. Patient-reported symptoms defined in the MPN-SAF TSS were present in a small minority (9.5%) of patients per arm at baseline (up to Week 4 of treatment) in this early-stage PV population. Occurrence of the defined symptoms remained low over long-term treatment, reported in 15.7% of patients in the ropeginterferon alfa-2b arm and 20.7% in the control arm during the 6th year of treatment. No phlebotomies were required to maintain hematocrit <45% in the 6th year of treatment in 81.4% of patients receiving ropeginterferon alfa-2b compared with 60.0% of patients in the control arm (p=0.005). Depletion of the JAK2V617F alle burden, which may lower the risk of progression to myelofibrosis, was observed in ropeginterferon alfa-2b treated patients; JAK2V617F allele burden <1% at 6 years was achieved in 19/92 (20.7%) patients in the ropeginterferon alfa-2b arm with baseline allele burden >10%. One patient met this threshold in the control arm (1/70 [1.4%]; p=0.0001). Event-free survival (risk events: disease progression, death and thromboembolic events) over ≥6 years of treatment was significantly higher among ropeginterferon alfa-2b treated patients than the control group (risk events reported in 5/95 vs. 12/74 patients, respectively; p=0.04 [Log-Rank]; Fig 1). Image:Summary/Conclusion: Long-term ropeginterferon alfa-2b therapy fulfils treatment goals important to patients with PV: a good quality of life as indicated by a low symptom burden and phlebotomy requirement, the potential to influence myelofibrosis risk, and better event-free survival versus BAT.

Interferon alfa not only restores normal blood cell counts in patients with polycythemia vera (PV) but can diminish the mutant JAK2V617F allele burden [1][2][3].After discontinuing long-term interferon therapy, hematologic responses may persist [4,5], which is more likely in patients achieving JAK2V617F allele burden <10% before stopping treatment [6].Allele burden declines gradually during interferon treatment [7]; however, no data from large, prospective, clinical studies of long-term treatment with pegylated alfa interferons are available.Results from 3 years' treatment in the phase 3, open-label, randomized clinical trial PROUD-PV and its phase 3b extension trial CONTINUATION-PV [8] led to regulatory approval of ropeginterferon alfa-2b (BESREMi®), a novel, monopegylated interferon alfa-2b with an extended administration interval of 2-4 weeks, in the European Union.The compound was subsequently approved by the US FDA for first-line treatment of PV.Here we report hematologic and molecular responses and safety results after 5 years' treatment in PROUD-PV and CONTINUATION-PV, which compared the efficacy and safety of ropeginterferon alfa-2b with hydroxyurea in the first year, and with best available treatment (BAT) thereafter.Study design and methods were published previously [8]; selection criteria, dosing and endpoints are described in the supplement (Supplementary Tables 1-3 and Supplementary Fig. 1).JAK2V617F-positive patients with PV who were hydroxyurea naïve or hydroxyurea pre-treated for <3 years without complete response, resistance or intolerance were randomized 1:1 (stratified by age, history of thromboembolic events and hydroxyurea pretreatment) in PROUD-PV to receive ropeginterferon alfa-2b or hydroxyurea for 12 months.Dosing increased until blood counts

Myeloproliferative neoplasms (MPN) are chronic stem cell disorders characterized by enhanced proliferation of myeloid cells, immune deregulation, and drug resistance. JAK2 somatic mutations drive the disease in 50–60% and CALR mutations in 25–30% of cases. Published data suggest that JAK2 -V617F-mutated MPN cells express the resistance-related checkpoint PD-L1. By applying RNA-sequencing on granulocytes of 113 MPN patients, we demonstrate that PD-L1 expression is highest among polycythemia vera patients and that PD-L1 expression correlates with JAK2 -V617F mutational burden ( R  = 0.52; p  < .0001). Single nucleotide polymorphism (SNP) arrays showed that chromosome 9p uniparental disomy (UPD) covers both PD-L1 and JAK2 in all MPN patients examined. MPN cells in JAK2 -V617F-positive patients expressed higher levels of PD-L1 if 9p UPD was present compared to when it was absent ( p  < .0001). Moreover, haplotype-based association analyses provided evidence for germline genetic factors at PD-L1 locus contributing to MPN susceptibility independently of the previously described GGCC risk haplotype. We also found that PD-L1 is highly expressed on putative CD34 + CD38 − disease-initiating neoplastic stem cells (NSC) in both JAK2 and CALR -mutated MPN. PD-L1 overexpression decreased upon exposure to JAK2 blockers and BRD4-targeting agents, suggesting a role for JAK2-STAT5-signaling and BRD4 in PD-L1 expression. Whether targeting of PD-L1 can overcome NSC resistance in MPN remains to be elucidated in forthcoming studies.

Myeloproliferative neoplasms (MPNs) are a group of acquired hematopoietic stem cell (HSC) disorders driven by mutations that constitutively activate physiologic signal transduction pathways essential for hematopoiesis. Most patients with classical MPNs harbor mutations within the Janus activated kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor (MPL) genes. The occurrence of driver mutations was thought to be mutually exclusive, but double-positive cases have been reported. However, in rare cases where clonal analysis was performed both mutations occurred in independent clones, suggesting mutual exclusivity at the single cell level.1-3 Recently, a single report described one patient who acquired a JAK2 mutation in a CALR mutant clone.4 Although the JAK2 mutation could be detected in HSCs as well as myeloid progenitors, the variant allele frequency was lowest in the HSC population. Although this report suggests that JAK2-V617F and CALR-del52 mutations can occur in a single clone, the effect on HSC fitness remains elusive. In this study, we tested the hypothesis that Jak2-V617F and Calr-del52 mutations are synthetic lethal if they occur in the same HSC. Since synthetic lethality at the HSC level can manifest as loss of competitive fitness over time or cell death, the only means by which such genetic interaction can be detected is using primary genetically engineered HSCs. Therefore, we generated a mouse model in which both mutations are co-expressed in the hematopoietic system, which allowed us to assess the phenotype of such animals and enabled evaluation of HSC fitness in transplantation experiments. We generated conditional knock-in mice that co-express Jak2 and Calr mutations in the presence of Cre recombinase in hematopoietic lineages (vav-iCre) and analyzed their phenotype (Figure 1A). C57BL/6 vav-iCre and Ly5.1/CD45.1 mice were purchased from Charles River Laboratories. Mice carrying Jak2-V617F mutation were previously published.5 and the Calr-del52 transgenic mice were generated in our laboratory.6 The Calr-del52 vav-iCre mice were further bred with Jak2-V617F mutant mice to generate mice expressing both mutations in the hematopoietic compartment. Blood parameters were measured on hematology analyzer scil Vet abc™ (Horiba ABX, Montpellier, France). Single cell suspensions of bone marrow, spleen, and blood were stained, with relevant panels of antibodies (Tables S1–S3), measured at BD LSRFortessa™ (Flow Cytometry - Core Facilities, Medical University of Vienna, Vienna, Austria) and analyzed using FlowJo software (Version 10.7.1; Ashland, Oregon, USA). Double positive offspring were born at expected Mendelian frequency comparable to the single positive littermates, suggesting no signs of synthetic lethality in utero (Figure 1B). The phenotype of the Calr/Jak2 double positive mice was significantly more severe compared to the single mutant mice. Notably, double positive mice showed more pronounced splenomegaly and higher platelet, leukocyte, granulocyte, monocyte, and lymphocyte count in peripheral blood compared to non-mutated or single mutated mice (Figures 1C–G and S1a). Hematocrit, red blood cell count, and hemoglobin were increased compared to non-mutated and Calr mice but did not exceed the values of Jak2 mice (Figures 1H and S1b,c). In line with the thrombocytosis, double mutant mice showed an increased frequency of megakaryocytes in the bone marrow (Figure 1I). Flow cytometry analysis of the blood and spleen revealed an altered cellular composition with a shift toward myeloid lineages (Figures S1d–i and S2a–f). While the stem cell frequency in double mutant mice seemed unaltered, the myeloid progenitor compartment showed a significant expansion of different progenitor types further explaining the excessive production of terminally differentiated myeloid cells (Figures S2g–j and S3a–i). Spleen and bone marrow of double positive mice also showed morphological changes. More obscured follicular architecture and enhanced extramedullary hematopoiesis was detected in the spleen. The bone marrow presented with more prominent megakaryocyte dyspoiesis and altered myeloid to erythroid ratios with no signs of myelofibrosis (Figure S4, reticulin staining not shown). Most importantly, the aggravated myeloproliferative phenotype of double positive mice manifested in lower overall survival compared to the Jak2 and Calr single mutated mice (Figure 1J). Our results are further supported by the report of a double mutant MPN patient who evolved from essential thrombocythemia to advanced myelofibrosis after acquisition of the JAK2-V617F mutation in a CALR-del52 mutated clone.4 To gain further insight into the cellular processes affected by Calr/Jak2 double mutation we compared gene expression of single and double mutated LSK (Lin- Sca-1+ c-Kit+) cells. While double mutated cells showed a very similar gene expression signature as the Jak2 single mutated cells, differential expression in comparison to Calr mutated cells was more pronounced (Figure S5a–d). Gene sets upregulated in double mutated versus Calr single mutated cells, such as coagulation, IL-2/STAT5 signaling, and IL-6/JAK/STAT3 signaling, are consistent with the increased disease phenotype. Although apoptosis is also among the enriched gene sets, the two genes clustering in this set, Btg2, and Ltb are also associated with maintenance of hematopoietic stem cells. Among the downregulated genes, three were associated with mTORC1 and hedgehog signaling. Compared to Jak2 single mutated cells, significantly downregulated genes clustered in heme metabolism and TNF-alpha signaling. Both genes categorized into the pancreas beta cell pathway, Mafb and Pklr are also involved in erythrocyte differentiation and metabolism. High expression of Ebi3 and STAT5 induced genes, that is Socs2 and Cish in the double mutant cells suggest that phenotypic differences are driven by JAK/STAT signaling (Figure S5e). To examine the impact of double mutation on HSC fitness, we performed competitive primary and secondary bone marrow transplantation (Figure S6a). Double positive bone marrow engrafted into recipients equally well as single positive cells suggesting no functional defect at the HSC level. We observed outgrowth of mutant cells in blood, spleen, and bone marrow with most prominent expansion of mutant cells in the myeloid compartment (Figures S6b–c and S7). Transplanted mice also developed an MPN phenotype reminiscent of the one observed in the transgenic animals (Figures S6d–f and S8). To further test if the double positive HSCs maintained their self-renewal capacity over time, we performed secondary transplantation. Double mutant HSCs efficiently engrafted in secondary recipients and mice developed peripheral blood changes indicative of myeloproliferative disease, further suggesting the absence of any long-term defect (Figures S6g, S9 and S10). Thus, the presence of both mutations in HSCs is unlikely to cause synthetic loss of HSC fitness. To date, with a single case being reported, there is no sizable JAK2/CALR double mutated patient cohort available to evaluate the disease phenotype and estimate survival. Our mouse model allowed phenotypic assessment of JAK2/CALR double mutated MPN. We observed a severely aggravated phenotype in double mutated compared to single mutated animals with increased hematocrit similar to Jak2 mutated mice and aggravated megakaryopoiesis and myelopoiesis compared to all other groups. Likewise, the reported patient also progressed to myelofibrosis after acquisition of the second mutation. Therefore, we conclude that the co-occurrence of both mutations might be a negative predictive indicator.4 HSCs did not show any signs of reduced fitness, ruling out mutual exclusivity due to synthetic lethality. Transplantation experiments using Calr-del52 or Jak2-V617F single mutated knock-in mice have been performed in previous studies and are in line with our results. While Jak2 mutant cells were reported to outgrow wt competitors, heterozygous Calr mutated HSCs did not show a significant proliferative advantage compared to wt cells.6-8 In this study we observed a steady increase in myeloid peripheral blood chimerism in all groups with little expansion in the Calr group and strongest expansion in mice that received double mutant bone marrow. The results were similar on the HSC level. In the Jak2 and double mutated group two mice each reached full mutant HSC chimerism in the secondary transplant. In short, double mutant HSCs engrafted equally well or better than their single mutant equivalents. Therefore, we can rule out reduced stem cell fitness as a reason for rare occurrence of double mutant patients. We hypothesize that patients carrying both mutations in one clone might be underdiagnosed as the most common diagnostic routine is single gene analysis for driver mutations in JAK2, CALR, and MPL at diagnosis. In the reported case, a JAK2-V617F mutation was acquired in a CALRdel52 mutated clone 18 years post initial diagnosis.4 This shows that a second driver mutation can be acquired decades later, which might be another factor leading to such patients being undetected since driver mutations are rarely re-analyzed after initial molecular diagnostics. In addition, establishing if the two mutations are monoclonal or bi-clonal, requires an in vitro colony assay. Presence of colonies with both mutations is the formal proof of monoclonal acquisition of the two mutations. However, this procedure is very time consuming and therefore rarely performed. Andrea Majoros and Robert Kralovics designed the study; Christina M Schueller, Andrea Majoros, and Harini Nivarthi performed experiments and collected the data; Christina M Schueller and Andrea Majoros analyzed the data; Christina M Schueller, Andrea Majoros, and Robert Kralovics wrote the manuscript. This study was supported by the Austrian Science Fund, FWF P34451-B granted to Robert Kralovics and P30041-B26 granted to Harini Nivarthi as well as the Austrian Academy of Sciences, DOC fellowship 26008 granted to Christina M Schueller. We thank Jakob Weinzierl and Elisabeth Fuchs for their technical assistance. We also thank Agnieszka Piszczek, Tamara Engelmaier, Julia Klughofer, and Mihaela Zeba for tissue processing and histochemical staining and Anoop Kavirayani for histopathological assessment. Moreover, we thank the CeMM Biomedical Sequencing Facility and Michael Schuster for performing RNA sequencing and data processing. This study was supported by the Austrian Science Fund, FWF P34451-B granted to Robert Kralovics and P30041-B26 granted to Harini Nivarthi as well as the Austrian Academy of Sciences, DOC fellowship 26008 granted to Christina M Schueller. The authors declare that there is no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Table S1. List of antibodies used in flow cytometry staining. Table S2. Flow cytometry staining panels for end point analysis of transgenic mice. Single cell suspensions from spleen and bone marrow were stained with two and three different panels, respectively. Table S3. Flow cytometry staining panels for endpoint analysis of bone marrow transplantation experiments. Single cell suspensions from spleen and bone marrow were stained with two and three different panels, respectively. Figure S1. Calr/Jak2 double mutated mice have altered blood composition. The blood composition of 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was compared. (a)–(c) Lymphocyte (a), erythrocyte count (b), and hemoglobin (c) were quantified using an automated blood counter. (d)–(i) Percentage of CD19+ B cells (d), CD4+ T cells (e), CD8+ T cells (f), GR-1+ granulocytes (g), CD11b + myeloid cells (h) and CD49b + NK (i) cells among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S2. Calr/Jak2 double mutated mice have altered spleen composition. Cellular composition of spleens from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was analyzed by flow cytometry. Percentage of CD19+ B cells (a), CD4+ T cells (b), CD8+ T cells (c), GR-1+ granulocytes (d), CD11b + myeloid cells (e) and CD49b + NK cells (f), Lin- Sca-1+ c-Kit+ LSK cells (g), Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC, h), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC, i) and Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP, j) among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S3. Calr/Jak2 double mutated mice have increased frequency of multipotent and myeloid progenitors in the bone marrow. Cellular composition of bone marrow from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2) was analyzed by flow cytometry. (a)–(d) Quantification of stem cells and multipotent progenitors among all viable cells: Lin- Sca-1+ c-Kit+ LSK cells (a), Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC, b), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC, c), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP, d). (e)–(g) Quantification of myeloid progenitors (Lin- Sca-1 low c-Kit+) and further stratification into FcRg+ CD34+ common myeloid progenitors (CMP, f), FcRg+ CD34+ granulocyte- monocyte progenitors (GMP, g) and FcRg- CD34- megakaryocyte-erythroid progenitors (MEP, h). (i) Percentage of CD71+ Ter-119 intermediate erythroid progenitors (ProE) among all viable cells. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S4. Calr/Jak2 mice have altered bone marrow and spleen morphology. Sternum (a) and spleen (b) from 8- to 9-week-old mice of different genotypes, wt vav-iCre (wt), Calr-del52fl/+ vav-iCre (Calr), Jak2-V617Ffl/+ vav-iCre (Jak2) and Jak2-V617Ffl/+/Calr-del52fl/+ vav-iCre (Calr/Jak2). After harvesting, organs were placed in Roti®Histofix 4% (Roth) over night at room temperature and moved to 1% Roti®Histofix in PBS for further storage at 4°C. Tissue processing, H&E staining, and pathological assessment were performed by Histopathology department at Vienna Biocenter Core Facilities. Figure S5. Double mutant cells show upregulation in the JAK/STAT signaling pathway. We analyzed the gene expression signature of sorted LSK (Lin- Sca-1+ c-Kit+) cells of the different genotypes. Differentially expressed genes with Log2 fold change ≥1.5 or ≤−1.5 and adjusted p-value ≤0.05 were considered as relevant. (a)–(d) Double mutant cells were compared to the single mutants. Gene set enrichment analysis was performed using Enrichr and genes were compared to the MSigDB Hallmark 2020 database. Individual genes falling in the different gene sets were highlighted in the corresponding color of their bar. (e) Expression of representative genes for the IL-2/STAT5 and IL-6/JAK/STAT3 pathways. Bars represent mean values of two sequencing reactions from one mouse and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. Figure S6. Double mutant stem cells retain their differentiation potential in primary and secondary bone marrow transplants. (a) Schematic representation of primary and secondary transplantation experiments. (b) Peripheral blood chimerism in myeloid cells was determined by FACS analysis every 4–5 weeks. (c) Terminal analysis of the bone marrow chimerism by FACS 25 weeks post first transplant. Due to lower survival only two and three of originally five mice could be included in this analysis for Jak2 and Calr/Jak2 groups, respectively. LSK – Lin- Sca-1+ c-Kit-; LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-); MPP – multipotent progenitors (Lin- Sca-1+ c-Kit- CD150- CD48+), MK – megakaryocytes (c-Kit- CD41+). (d)–(f) Mice which received the primary competitive bone marrow transplant were observed for phenotypic changes in peripheral blood using an automated blood counter every 4–5 weeks. (g) FACS quantification of LT-HSC chimerism of donor mice (primary transplant, 1st) and recipient mice 13 weeks post secondary transplant (2nd). LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S7. Calr/Jak2 cells are dominant in the myeloid compartment in blood and spleen and provide the majority of myeloid progenitors in the bone marrow. FACS analysis of chimerism in blood, spleen, and bone marrow at the endpoint of the primary transplantation experiment (25 weeks). (a) Chimerism in peripheral blood cells was determined: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD49b + NK cells and CD3+ T cells. (b) Chimerism in splenocytes was determined: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD41+ megakaryocytes (MK), and CD3+ T cells. (c) Chimerism in splenic hematopoietic stem and progenitor populations was determined: Lin- Sca-1+ c-Kit+ LSK cells, Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP). (d) Chimerism in short term hematopoietic stem cells (ST-HSC; Lin- Sca-1+ c-Kit+ CD48- CD150) and myeloid progenitor subpopulations (FcRg+ CD34+ common myeloid progenitors (CMP), FcRg- CD34- megakaryocyte-erythroid progenitors (MEP) and FcRg+ CD34+ granulocyte-monocyte progenitors (GMP)) in the bone marrow was determined. The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). Figure S8. Transplanted mice developed an MPN phenotype with peripheral blood changes similar to the transgenic animals. (a)–(h) Blood parameters from 24-week-old mice (the latest time point with 4 mice/group alive) from the primary bone marrow transplantation experiment were analyzed using an automated blood counter. (i) Comparison of spleen size (spleen/body weight) at the endpoint of the primary transplantation experiment (25 weeks). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. Figure S9. Calr/Jak2 cells showed persistence in blood, spleen, and bone marrow after secondary transplantation. FACS analysis of chimerism in blood, spleen, and bone marrow at the at the endpoint of the secondary transplantation experiment (13 weeks). (a) Chimerism in peripheral blood: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD49b + NK cells and CD3+ T cells. (b) Chimerism in splenocytes: CD19+ B cells, GR-1+ granulocytes, CD11b + myeloid cells, CD41+ megakaryocytes (MK) and CD3+ T cells. (c) Chimerism in splenic hematopoietic stem and progenitor populations: Lin- Sca-1+ c-Kit+ LSK cells, Lin- Sca-1+ c-Kit+ CD48- CD150+ long term hematopoietic stem cells (LT-HSC), Lin- Sca-1+ c-Kit+ CD48- CD150- short term hematopoietic stem cells (ST-HSC), Lin- Sca-1+ c-Kit+ CD48+ CD150- multipotent progenitors (MPP). (d) Chimerism in different bone marrow populations: LSK – Lin- Sca-1+ c-Kit-; LT-HSC – long term hematopoietic stem cells (Lin- Sca-1+ c-Kit- CD150+ CD48-); ST-HSC – short term hematopoietic stem cells (Lin- Sca-1+ c-Kit+ CD48- CD150), MPP – multipotent progenitors (Lin- Sca-1+ c-Kit- CD150- CD48+), MK – megakaryocytes (c-Kit- CD41+), and myeloid progenitor subpopulations (FcRg+ CD34+ common myeloid progenitors (CMP), FcRg- CD34- megakaryocyte-erythroid progenitors (MEP) and FcRg+ CD34+ granulocyte- monocyte progenitors (GMP)). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM). Figure S10. Mice showed peripheral blood changes indicative of myeloproliferative disease. Terminal analysis of 13-week-old mice which received the secondary transplant. (a)–(h) Blood parameters were analyzed using an automated blood counter. (i) Comparison of spleen size (spleen/body weight). The dots represent values for individual animals; Lines represent mean values for the group and the error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001); the p-values were calculated using ordinary one-way ANOVA test. The gray area between dotted lines represents physiological levels for wt C57BL/6 mice. 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Introduction: Patients with "low-risk” (LR) PV are managed conservatively despite higher risk of vascular events and impaired quality of life compared to a population without PV. Acknowledging new evidence (LOW-PV study, Barbui et al 2021), recent treatment guidelines (European LeukemiaNet [ELN] 2021) recommend ropeginterferon alfa-2b (BESREMi®) as a cytoreductive treatment in LR PV patients meeting specific criteria as well as in high-risk (HR) PV patients. Final data from the phase 3 PROUD-PV and CONTINUATION-PV trials (NCT01949805; NCT02218047) demonstrated the long-term efficacy and safety of ropeginterferon alfa-2b compared with hydroxyurea/best available treatment (HU/BAT); here we report results in the ropeginterferon alfa-2b arm by risk stratum. Methods: PV patients were randomized 1:1 and received ropeginterferon alfa-2b or HU/BAT for ≥6 years. In the ropeginterferon alfa-2b arm, subgroup analyses were performed post-hoc by risk stratum (LR: age ≤60 years and no history of thrombosis; all others HR). Complete hematologic response (CHemR) and molecular response defined by modified ELN criteria were assessed in the full analysis set for CONTINUATION-PV (N=95); safety analyses included all data regardless of roll-over (N=127). Results: CONTINUATION-PV enrolled 46 LR and 49 HR patients in the ropeginterferon alfa-2b arm. At baseline, mean age was 50.7 vs 63.7 years respectively, and median duration since PV diagnosis was 1.9 months vs 1.5 months. HR patients had higher platelet and leukocyte counts at baseline and more frequently presented with clinically significant splenomegaly or PV-related symptoms. Median JAK2V617F allele burden was similar for LR and HR patients (37.0% vs 39.4%), however LR patients were less likely to harbor additional non-driver mutations (11.4% vs 24.4%). The median 4-weekly ropeginterferon alfa-2b dose was comparable in LR and HR patients throughout the study (in the 6th year, 499 µg and 489µg respectively) as was the use of an extended 3-4 weekly dose regimen (6th year: 59.5% vs 65.4%). LR patients achieved a significantly higher CHemR rate than HR patients during long-term treatment (Month 72: 73.2% vs 38.3% in LR and HR respectively [RR: 2.41; 95% CI: 1.50 to 3.90; p=0.0003]). Higher CHemR among LR patients was confirmed in a sensitivity analysis including discontinued patients (last observation carried forward [LOCF]; 80.4% in LR vs 65.3% in HR patients at Month 72) indicating that in the HR group, more patients discontinued despite CHemR. In general, discontinued patients were older than those who completed the study (mean age 62.8 years vs 55.2 years) but did not have a longer duration of PV or markedly greater symptom burden at baseline. Importantly, regardless of risk stratum, most patients receiving ropeginterferon alfa-2b did not require any phlebotomy for the entire 6th year (85.7% of LR patients and 75.0% of HR patients entering the 6th year [p=0.3]). LR patients had a higher molecular response rate than HR patients (LOCF at Month 72: 84.4% vs 49.0% respectively, RR: 2.15 [95% CI: 1.37 to 3.37]; p=0.0009) and achieved molecular response more rapidly (median time to first response: 12 months [95% CI: 12 to 18] vs 18 months [95% CI: 12 to 24]; Log-rank test p=0.03). The median JAK2V617F allele burden was 5.6% for LR and 17.9% for HR patients at Month 72 (p<0.0001). During the entire PROUD-PV/CONTINUATION-PV studies (≥6 years exposure), adverse drug reactions (ADRs) to ropeginterferon alfa-2b led to withdrawal of treatment in 1 (1.8%) LR patient (due to sarcoidosis), and in 13 (18.3%) HR patients (due to thrombocytopenia [n=1], elevated transaminases [n=1], psychiatric disorders [n=2], autoimmune disorders [n=6], hypothyroidism [n=2] and dyspnea/pneumonitis [n=1]). LR patients were also less likely than HR patients to experience ADRS of grade ≥3 severity (5.4% vs 23.9%). Conclusion: These results demonstrate the benefit of ropeginterferon alfa-2b therapy in both HR and LR patient populations. LR patients may have a greater potential benefit: higher hematologic and molecular response rates can be safely achieved, and the patients are more likely to remain on long-term treatment. These data provide further evidence for an early treatment start as recently outlined by the updated ELN guidelines.

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P<.01) or HD (P<.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p<.005) and the same was observed in PMF (p<.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P<.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P<.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p<.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p<.001 and P<.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P<.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.