While TP53 mutations in myeloproliferative neoplasms (MPN) are associated with an increased risk of leukemic transformation, not all patients carrying a TP53 mutation progress. To better risk-stratify MPN patients with TP53 mutations, we analyzed data from 1540 patients treated at four specialized cancer centers. Among them, 1429 had wildtype TP53 and 111 had mutations in the TP53 gene. At first MPN diagnosis, 32% had polycythemia vera, 39% had essential thrombocythemia, and 25% had primary myelofibrosis. Among all MPN patients with TP53 mutations, presence of fibrosis in the bone marrow (hazard ratio (HR): 3.84, 95% CI: 1.98-7.43), multi-hit TP53 mutation status (HR: 2.74, 95% confidence interval (CI): 1.52-4.97), and higher PHANTM score (HR: 1.87, 95% CI: 1.02-3.42) were associated with worse OS in a multivariable analysis. Based on these variables, we developed a risk model to identify TP53-mutated MPN patients who are at high risk for inferior OS. Median OS from time of TP53 detection was 0.5 years in high-risk patients, compared to 2.3 years for patients with intermediate risk and 6.3 years for patients with low risk. This scoring system may help refine risk stratification for chronic phase MPN patients harboring TP53 aberrations.

The discovery of calreticulin (CALR) mutations in patients with myeloproliferative neoplasms (MPN) has paved the way for the elucidation of a unique disease mechanism that is particularly well-suited to targeting by biologics. All MPN-associated pathogenic CALR mutations are characterized by a frameshift, resulting in translation of the same neoantigen peptide. This neoantigen directly activates the thrombopoietin receptor, leading to uncontrolled neoplastic cell proliferation. Current therapeutic approaches for MPN are focused primarily on blood count control. Furthermore, current approaches are neither disease-modifying nor clonally selective. However, as the mutant CALR neoantigen peptide is functional and not expressed in normal physiology, it is an ideal drug target. Here, we review the structure and function of mutant CALR, including the subtle yet clinically and therapeutically relevant differences between the two most commonly occurring types of mutation. We also review the current therapeutic landscape for CALR-mutated MPN, highlighting the areas in which current approaches are inadequate. Finally, we review ongoing clinical and preclinical experimental approaches for targeting mutant CALR in MPN in a clonally selective manner using monoclonal antibodies, bispecific antibodies, cancer vaccination, chimeric antigen receptor T cells, and antibody-drug conjugates. Taken together, we expect that ongoing developments in mutant CALR-targeted therapeutics will lead to promising novel strategies for long-term disease control.

Somatic mutations in RNA splicing regulators, including the serine/arginine-rich protein SRSF2, are frequently observed in myeloid malignancies. Using mouse models and primary human samples, we investigated the impact of SRSF2 mutations on erythropoiesis. We found reduced erythropoiesis in Srsf2P95H versus wild-type mice upon stress-induced erythropoiesis and identified that SRSF2 mutations correlate with reduced hemoglobin in JAK2-mutant patients with myeloproliferative neoplasms (MPN). Consistent with this, Jak2V617F-Srsf2P95H versus Jak2V617F mice displayed reduced red blood cell counts and erythroid precursor frequencies. RNA-sequencing on erythroid precursors showed reduced expression of heme metabolism and mitotic spindle-related genes, and increased expression of mTORC1 signaling in Srsf2P95H versus wild-type cells. RNA splicing analyses on the same cells and on human patient samples identified aberrant FYN splicing in SRSF2mut cells, with increased aberrant FYNB over normal FYNT transcripts. FYNB, but not FYNT, expression resulted in reduced erythroid differentiation and increased phosphorylation of mTORC1 downstream target S6. Additionally, increased S6 phosphorylation was confirmed in primary Srsf2P95H erythroid cells. mTORC1 pathway inhibition using rapamycin normalized FYNB- and Srsf2P95H-induced impaired erythropoiesis and significantly increased erythroid colony formation of SRSF2-mutant myelodysplastic neoplasm (MDS) bone marrow cells. Our data reveal targetable molecular mechanisms of impaired erythropoiesis in SRSF2-mutant cells.

More than a decade following the discovery of Calreticulin (CALR) mutations as drivers of myeloproliferative neoplasms (MPN), advances in the understanding of CALR-mutant MPN continue to emerge. Here, we summarize recent advances in mehanistic understanding and in targeted therapies for CALR-mutant MPN. Structural insights revealed that the mutant CALR-MPL complex is a tetramer and the mutant CALR C-terminus is exposed on the cell surface. Targeting mutant CALR utilizing antibodies is the leading therapeutic approach, while mutant CALR-directed vaccines are also in early clinical trials. Additionally, chimeric antigen receptor (CAR) T-cells directed against mutant CALR are under evaluation in preclinical models. Approaches addressing the cellular effects of mutant CALR beyond MPL-JAK-STAT activation, such as targeting the unfolded protein response, proteasome, and N-glycosylation pathways, have been tested in preclinical models. In CALR-mutant MPN, the path from discovery to mechanistic understanding to direct therapeutic targeting has advanced rapidly. The longer-term goal remains clonally-selective therapies that modify the disease course in patients.

Over the course of the last decade, genomic studies in the context of normal human hematopoiesis have provided new insights into the early pathogenesis of myeloproliferative neoplasms (MPN). A preclinical phase of MPN, termed clonal hematopoiesis was identified and subsequent lineage tracing studies revealed a multi-decade long time interval from acquisition of an MPN phenotypic driver mutation in a hematopoietic stem cell to the development of overt MPN. Multiple germline variants associated with MPN risk have been identified through genome-wide association studies and in some cases functional interrogation of the impact of the variant has uncovered new insights into hematopoietic stem cell biology and MPN development. Increasingly sophisticated methods to study clonal contributions to human hematopoiesis and measure hematopoietic stem cell fitness have helped to discern the biology underlying the tremendous clinical heterogeneity observed in MPN. Despite these advances, significant knowledge gaps remain, particularly with respect to germline genetic contributors to both MPN pathogenesis and phenotypic diversity, as well as limitations in the ability to prospectively quantify rates of clonal expansion in individual MPN patients. Ultimately, we envisage a personalized approach to MPN care in the future, in which an individualized genetic assessment can predict MPN trajectory and this information will be used to inform and guide therapy. MPN is particularly amenable to precision medicine strategies and our increased understanding of the evolution of MPN from normal blood stem cells provides a unique opportunity for early therapeutic intervention approaches and potentially MPN prevention strategies.

Introduction: Although the inferred “fitness” of clonal hematopoesis (CH) driver mutations differs depending on the gene mutation, the impact of environmental factors that may promote or impair CH expansion remain largely unknown. We hypothesized that obesity influences the clonal expansion rate of common CH driver mutations. Methods: To test this hypothesis, we first interrogated data from the UKBiobank (UKBB) (n=425,573 exomes) to evaluate the relationship between body mass index (BMI) (kg/m2) and four common CH mutations, namely DNMT3A, TET2, ASXL1, and JAK2. For functional studies we focused on JAK2V617F, using a novel Fgd5-CreER-Jak2V617F mouse model we generated. In this model, Jak2V617F expression is induced in a small percentage of long-term (LT)-HSC which are tracked sequentially in primary mice in an unirradiated bone marrow niche. To quantify Jak2V617F expression in the model, we developed a digital droplet PCR assay. We also generated a chimeric bone marrow transplant Jak2V617F model and performed bulk RNA-sequencing (RNAseq) on purified lineagelow Sca-1+ c-kithigh (LSK) cells expressing Jak2V617F or wild-type Jak2, isolated from the same mouse. Finally, we performed single-cell RNAseq on Jak2V617F-expressing c-kithigh cells purified from chimeric transplant mice. Mice fed an obesity diet received 60 kcal% fat while mice fed a control diet received 10 kcal% fat. Results: In UKBB analyses, we identified genotype-specific patterns of association between BMI and the presence of CH mutations. JAK2 (OR 0.51; 95% CI 0.27-0.96; p=0.036) and DNMT3A mutations (OR 0.92; 95% CI 0.87-0.97; p=0.0028) were negatively associated with a BMI >30 relative to BMI <25, whereas mutations in ASXL1(OR 1.40; 95% CI 1.23-1.60; p=8.8x10-7) and TET2 (OR 1.16; 95% CI 1.04-1.31; p=0.0095) were positively associated with BMI >30. To further explore the negative association between JAK2V617F and obesity, we generated a cohort of Fgd5-CreER-Jak2V617F mice and fed half the mice an obesity diet and the other half a control diet. After 24 weeks, Jak2-mutant mice fed the control diet developed a significantly higher hematocrit (HCT) as compared to Jak2-mutant mice fed the obesity diet (p=0.0043). The HCT remained significantly higher in control mice as compared to obese mice over a 46-week period (p<0.0001). Strikingly at 46 weeks, 5/7 mice (71%) fed the control diet showed signs of MPN as compared with 0/6 mice (0%) fed the obesity diet. Furthermore, overall survival was significantly improved in Jak2-mutant obese mice as compared to Jak2-mutant mice fed a control diet (p=0.03). Subsequently, after the death of most control mice, one Jak2-mutant obese mouse developed an elevated HCT, indicating that obesity prolonged the latency to MPN development in Jak2V617F mice. In the chimeric transplant model, gene set enrichment analysis (GSEA) showed significantly increased interferon alpha (NES=2.55, FDR≈0), interferon gamma (NES=2.26, FDR≈0), and interleukin-6 (NES=2.15, FDR=5.46x10-05) signaling in Jak2-mutant LSK cells isolated from obese animals as compared to wild-type LSK cells, a finding not observed in control mice. Single-cell RNAseq experiments further validated these findings, where we identified interferon induced transmembrane protein 1 (IFITM1) as one of the most differentially upregulated genes in Jak2-mutant LT-HSCs from obese mice (adjusted p-value=1.08x10-54) as compared to wild-type LT-HSCs, and unlike Jak2-mutant LT-HSCs from control mice. Conclusions: To our knowledge, our study is the largest to date to evaluate the relationship between obesity and CH and the first to investigate the negative association between JAK2V617F CH and obesity, using functional studies. We developed a novel Fgd5-CreER-Jak2V617F mouse model, which allowed us to study the transition from CH to overt MPN in an unperturbed, non-irradiated bone marrow niche. Transcriptomic profiling points to activation of the interferon signaling pathway in Jak2-mutant HSC as a possible mechanism by which JAK2-mutant LT-HSC may preferentially exhaust during obesity. These findings support testing pegylated interferon as a potential treatment for individuals with JAK2-mutant CH and a clinical protocol is currently under development at our institution. Ongoing functional studies are investigating additional metabolic targets in LT-HSC with the goal of uncovering novel treatment approaches for JAK2-mutant CH.

Background: Essential thrombocythemia (ET) and polycythemia vera (PV) are myeloproliferative neoplasms (MPNs) characterized by increased risk of thrombotic events and progression to more aggressive leukemias. Risk stratification in ET and PV is based on thrombotic risk, and patients at high risk of thrombosis (i.e. patients >60 years of age, those with a prior history of thrombosis, and patients > 60 with a JAK2 mutation in ET) are often started on cytoreduction for thrombosis prevention. However, in addition to thrombotic risk, ET and PV patients experience significant symptoms due to activation of the JAK/STAT signaling pathway resulting in an increase in inflammatory cytokines. These symptoms include fatigue, itching, night sweats, bone pain, and brain fog. Symptoms occur in MPN patients regardless of thrombotic risk; in low-risk patients, many of whom are younger and of working age, symptoms can be severe and adversely impact functioning and quality of life. Ruxolitinib is a JAK1/2 inhibitor currently approved in myelofibrosis (MF) and as a second-line agent in PV. It has been demonstrated to significantly improve symptom burden based on its ability to ameliorate inflammation through JAK/STAT inhibition. Ruxolitinib is not approved in ET or as a first-line agent in PV. However, it is a rational therapy to address symptom control in ET and PV patients who have high symptom burden. The purpose of this trial is to evaluate the preliminary efficacy of ruxolitinib in improving symptom burden in ET and PV patients who are low risk for thrombosis but experience significant MPN-related symptoms. Study Design and Methods: This is a multi-center, non-randomized, two-stage phase 2 trial evaluating ruxolitinib in low-risk, symptomatic ET and PV patients (NCT04644211). Enrollment is currently open at Dana-Farber Cancer Institute, Massachusetts General Hospital, and Beth Israel Deaconess Medical Center. Ruxolitinib is dosed at 10 mg BID and dose-escalated to a maximum of 25 mg BID based on symptoms and tolerability. Eligible patients include adults (age >18) with a diagnosis of ET and PV by World Health Organization 2016 criteria. ET patients must be very low, low, or intermediate risk by Revised International Prognostic Score for Thrombosis (R-IPSET), and PV patients must be low risk by National Comprehensive Cancer Network (NCCN) criteria. Patients must have high symptom burden to be eligible, as defined by a baseline MPN Symptom Assessment Form (SAF) Total Symptom Score (TSS) >10 with at least one individual component of the MPN SAF TSS >5. Patients remain eligible if they have had prior cytoreduction for symptom control. The primary objective of this study is to evaluate ruxolitinib's efficacy in improving symptom burden as measured by the percentage of patients who achieve >50% improvement of baseline symptoms at week 12 MPN-SAF TSS score. Secondary objectives of this study are to evaluate the efficacy of ruxolitinib in achieving (1) hematologic remission, as defined by International Working Group (IWG) criteria; (2) change in percentage spleen volume by imaging, in patients with baseline splenomegaly; and (3) other quality of life metrics including the modified Patient-Reported Outcomes Measurement Information System (mPROMIS-57) and Patient Global Impression of Change (PGIC). Exploratory objectives include evaluation of ruxolitinib's effect on inflammatory cytokine production and its relationship with symptom burden. We will also perform next-generation-sequencing to evaluate the effect of ruxolitinib on driver and non-driver mutation burden. A total of 30 patients will be enrolled in a Simon two-stage design where the first 15 patients are assessed at week 12. If >3 responses occur in the first stage, an additional 15 patients will be enrolled. The study is considered promising if >6 responses are recorded in 30 patients. The first patient consented to the study in March 2022, and as of this writing 15 patients have enrolled (7 ET, 8 PV; median age 49.2 years; 13/15 female), with confirmed responses in at least 3 patients. The trial is therefore currently enrolling an additional 15 patients for Stage 2.

Background: Studies have shown an increased incidence of myeloid neoplasia (MN) in patients with sickle cell disease (SCD). It is hypothesized that increased erythropoietic stress may influence acquisition of molecular drivers of MN in SCD. However, present understanding of MN presentation and outcomes in SCD and other hereditary hemoglobinopathies and hemolytic anemias (HHAs) is limited to case reports. Systematic evaluation of MNs in patients with HHAs will better contextualize the risk of MN in this population relative to non-HHA controls. Methods: Patients with HHAs were identified from the Mass General Brigham Research Patient Data Repository using ICD-10 codes. Evaluated HHAs included sickle cell disease (SCD), sickle cell trait (SCT), thalassemia major, thalassemia trait, other hemoglobinopathy traits, and hereditary spherocytosis or elliptocytosis. A final HHA-MN cohort included patients with laboratory confirmed HHA diagnoses as well as incident myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), Ph+ chronic myelogenous leukemia (Ph+ CML), Ph negative myeloproliferative neoplasms (Ph- MPNs) or acute myeloid leukemia (AML). A control cohort of non-HHA MN patients was obtained from the Dana-Farber Hematologic Malignancies Data Repository. Clinical, molecular, and survival data were summarized and compared between HHA-MN and controls. Results: The HHA-MN cohort included 62 patients with HHAs and incident MN (Table 1). HHAs included SCD (n = 6, 9.7%), SCT (n = 9, 14.5%), thalassemia trait (n = 36, 58.1%), thalassemia major (n = 2, 3.2%), hereditary spherocytosis or elliptocytosis (n = 4, 6.5%), and other hemoglobinopathy traits (n = 5, 8.1%). MN was diagnosed at a median age of 61 years [IQR: 48, 70] in the HHA-MN cohort and incident MNs included AML (n = 12, 19.4%), MDS/CMML (n = 22, 35.4%), Ph+ CML (n = 6, 9.7%), and Ph- MPNs (n = 22, 35.4%). A control population of 3388 MN patients without HHA included cases of AML (n = 754, 22.2%), MDS/CMML (n= 1272, 37.5%), Ph+ CML (n = 354, 10.4%), and Ph- MPNs (n = 1008, 29.7%). Compared to HHA patients, control patients were older at MN diagnosis (median age 65 years [IQR: 56, 73], p = 0.0071), driven by significantly younger ages at AML diagnosis for HHA-MN patients (median age 50 years [IQR: 33, 63] vs 65 years [IQR: 57, 73], p =0.0044). AML was the most common MN in SCD (n = 4, 66.7%) whereas incident MDS/CMML was the predominant MN among individuals with thalassemia trait (n = 12, 33.3%) or hereditary spherocytosis and elliptocytosis (n = 2, 50%). Diagnostic cytogenetic data were available for 45 patients in the HHA-MN cohort (72.6% of cohort). Complex karyotype, defined as 3 or more chromosomal aberrations, was observed in 3 of 11 (27.3%) AML, 3 of 19 (15.8%) MDS/CMML and 1 of 17 (5.9%) Ph- MPN cases. Of the 33 HHA-MN patients with available diagnostic next generation sequencing, 60.6% had 2 or more pathogenic mutations at MN diagnosis. Including 2 cases of TP53-mutant AML in patients with SCD, the majority (90%) of AML and 38.9% of MDS/CMML diagnosed in HHA-MN patients were intermediate to adverse risk by ELN criteria or intermediate to very high risk by IPSS-R, respectively. High risk disease was also common in our control MN cohort, where 88.3% of AML cases were intermediate or adverse risk by ELN criteria and 55.9% of MDS/CMML cases were intermediate to very high risk by IPSS-R. Survival varied by subtype of HHA and MN (Figure 1). Survival was lowest for SCD (33.3% at 60-months) and highest for thalassemia traits (67.1% at 60 months). In HHA patients compared to controls, 60-month survival was 65.0% overall (vs 56.4%), 14.3% for AML (vs 23.8%), 51.0% for MDS/CMML (vs 43.9%), 90.5% in Ph- MPN (vs 80.6%), and 100% in Ph+ CML (vs 89.4%). Within the limitations of our sample size, survival differences were not statistically significant. Conclusion: In this retrospective analysis, we observe younger age of diagnosis of myeloid malignancy for patients with HHAs compared to non-HHA controls. Inferior survival in SCD and thalassemia major is largely explained by a higher proportion of AML diagnoses in patients with SCD. AML and MDS/CMML diagnosed in HHA patients tended to be intermediate or high-risk disease, with survival similar to the non-HHA control population that was also enriched for higher risk patients. Validation of our findings in an expanded cohort of HHA patients is currently underway.

Supplementary Figure S5. Immunoblotting of Calr-immunoprecipitated proteins and whole cell lysates from Ba/F3-Cas9 and Ba/F3-MPL-Cas9 cells targeted with Calr-directed CRISPR gene editing using a control guide, Calr guide m1, or Calr guide m2 demonstrates increased binding between MPL and mutant Calr (lane 3 - 4 (m1) and lane 5 -6 (m2)) as compared to wild type Calr (lane 2).

MPNs including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) are characterized by JAK2, CALR and MPL mutations. Additional mutations outside these driver genes also co-occur. To better understand interactions between phenotypic driver mutations and concomitant mutations, we evaluated patterns of somatic mutation acquisition in 1301 MPN (535 ET, 392 PV, 331 MF, and 43 MPN NOS) patients who underwent clinical next generation sequencing (NGS) at our institution. For this analysis, we focused on a single NGS panel obtained closest to MPN diagnosis. Median age at MPN diagnosis was 59 years, with MF patients older than ET/PV patients (p<4e-11). 67%, 17%, and 4.5% of patients had mutations in JAK2, CALR, or MPL. The average variant allele fraction (VAF) for JAK2, CALR and MPL was 48%, 34%, and 40%. 52% of patients had pathogenic mutations in an additional gene (range 0-7), most frequently TET2, DNMT3A, and ASXL1. PV patients were more likely to have a TET2 mutation compared with JAK2-mutated ET patients (42% vs 25%). Mutation combinations that occurred together more frequently than expected (p<1e-3) included DNMTA-TET2, ASXL1-TET2, ASXL1-DNMT3A, and ASXL1-SRSF2. We then investigated how these concomitant mutations interacted with the MPN driver mutation and each other. JAK2-mutatedMPN patients with no concomitant mutations were younger than JAK2-mutated patients with TET2 or ASXL1 mutations. In contrast, age distributions were similar between JAK2-only patients and those with JAK2 and DNMT3A mutations (Fig 1A). This observation held when looking within PV and ET, and whether patients with single or multiple concomitant mutations were considered. Since DNMT3A, TET2 and ASXL1 clonal hematopoiesis is associated with increasing age, the similar age distribution of JAK2-only and JAK2-DNMT3A co-mutant patients was an unexpected finding, and worthy of further study. We evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of age. The fraction of patients with TET2 and ASXL1 mutations increased with age, but not the fraction of patients with DNMT3A mutations (95% CI of slope: 4e-3 to 7e-3 for TET2, 0 to 3e-3 for DNMT3A). Similarly, when we evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of JAK2 VAF, the fraction of patients with TET2 and ASXL1 mutations increased as JAK2 VAF increased, but the fraction of patients with DNMT3A mutations decreased (Fig 1B; 95% CI of slope: 1e-3 to 3e-3 for TET2, -2e-3 to 0 for DNMT3A). One potential explanation for this is that JAK2 and DNMT3A mutations occur as independent clonal acquisitions and that the JAK2-mutant clone “out-competes” the DNMT3A-mutant clone over time. At JAK2 VAFs of 50% and 100%, the ratio of TET2:JAK2 VAFs clustered at 1.0 and 0.5, suggesting frequent presence of a dominant JAK2-TET2 clone in all cells with heterozygous or homozygous JAK2 mutations. ASXL1 mutations occurred mostly in patients with JAK2 VAF > 50%, consistent with it being a subclonal acquisition. In contrast, DNMT3A concomitant mutations were more likely to occur in patients with JAK2 VAF<50% vs >50%, again suggesting that DNMT3A clones diminish as JAK2 clones expand. 6/45 DNMT3A mutations that occurred as the sole concomitant mutation were R882 hotspot mutations. However, DNMT3A was more likely to co-occur at JAK2 VAF>50% in patients with >1 concomitant mutation (most commonly TET2). This suggests that in the context of JAK2-mutant driven MPNs, DNMT3A subclones may require the presence of an additional mutation to expand. In summary, we found DNMT3A mutations behave differently from TET2 and ASXL1 mutations in JAK2-mutant MPNs. Patients with JAK2-mutant MPN are less likely to have DNMT3A mutations as JAK2 VAF increases. Compared with TET2 or ASXL1, DNMT3A appears more likely to exist as an independent clone in JAK2-mutant MPN, with our data suggesting the JAK2-mutant clone exhibits increased fitness compared to the DNMT3A-mutantclone over time. However, DNMT3A mutations persist when accompanied by other concomitant mutations (particularly TET2). These results raise interesting questions regarding clonal competition in MPN. Since all our analyses were performed from single clinical NGS panels, our findings require additional validation, including with multi-gene single-cell genotyping, which we are currently pursuing.

Supplementary Figure S4. (A) Comparison between human and mouse wild-type (WT) calreticulin Cterminal tail. Blues indicates non-conserved regions between human and mouse. The wild-type proteins are highly conserved between the two species and contain many negatively-charged glutamic acid (E) amino acids. (B) Comparison between human and mouse amino acid sequences corresponding to the mutant C-terminal part of the human and mouse mutant with red shading indicating non-conserved regions. Despite the large number of mismatches, both mutant C-terminal tails are rich in positively charged amino acids (arginine (R) and lysine (K)).

Supplementary Table S1. Pre-ranked GSEA statistics between mutant and wild-type CALR 24 hours post-IL-3 withdrawal amongst STAT5, STAT3 and JAK2 inhibitor signatures from MSiGDB and literature.

Supplementary Figure S1. (A) Relative frequency of GFP positive cells in peripheral blood over time in BMT mice receiving empty vector, CALRWT or CALRMUT-expressing c-Kit+ BM cells. (B) Histopathologic H&E sections of BM from representative EV, CALRWT or CALRMUT recipient mice demonstrates megakaryocytic hyperplasia in CALRMUT recipient animals (100X magnification) (C) A N-terminal anti-CALR antibody demonstrated strong, diffuse cytoplasmic reactivity in megakaryocytes in representative normal and CALR-mutant patient bone marrow biopsies. In addition, cytoplasmic reactivity was noted in smaller mononuclear cells, which were morphologically most suggestive of early myeloid forms. Strong reactivity was not observed in mature erythrocytes or in mature granulocytes. (D) Flow cytometric analysis of Lin−Sca-1+c-Kit+ (LSK) and Lin−Sca-1−c-Kit+ (LK) cell numbers in the bone marrow of BMT mice receiving empty vector, CALRWT or CALRMUTexpressing BM cells 16 weeks post bone marrow transplantation demonstrates a significant increase in LSK cells in CALRMUT recipients. All p values were determined by unpaired two-tailed Student's t test. (*0.01 < p < 0.05; (**0.001 < p < 0.01; ns, not significant).

Supplementary Figure S2. Schema of CRISPR/Cas9 gene-editing targeting of exon 9 of Calr.

Mutations in calreticulin are one of the key disease-initiating mutations in myeloproliferative neoplasms (MPN). In MPN, mutant calreticulin translates with a novel C-terminus that leads to aberrant binding to the extracellular domain of the thrombopoietin receptor, MPL. This cell surface neoantigen has become an attractive target for immunological intervention. Here, we summarize recent advances in the development of mutant calreticulin targeting antibodies as a novel therapeutic approach in MPN.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription signaling. As a result, JAK inhibitors have been the standard therapy for treatment of patients with myelofibrosis (MF). Although currently approved JAK inhibitors successfully ameliorate MPN-related symptoms, they are not known to substantially alter the MF disease course. Similarly, in essential thrombocythemia and polycythemia vera, treatments are primarily aimed at reducing the risk of cardiovascular and thromboembolic complications, with a watchful waiting approach often used in patients who are considered to be at a lower risk for thrombosis. However, better understanding of MPN biology has led to the development of rationally designed therapies, with the goal of not only addressing disease complications but also potentially modifying disease course. We review the most recent data elucidating mechanisms of disease pathogenesis and highlight emerging therapies that target MPN on several biologic levels, including JAK2-mutant MPN stem cells, JAK and non-JAK signaling pathways, mutant calreticulin, and the inflammatory bone marrow microenvironment.

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by > 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p<0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

Introduction: The reported impact of gene mutations, including JAK2-V617F, other MPN phenotypic drivers and additional cooccurring mutations on outcomes after allogeneic transplantation (HCT) in myelofibrosis (MF) has been inconsistent. While the MTSS score incorporated ASXL1 and JAK2 (non MPL/CALR mutations) as unfavorable predictors after HCT, other studies reported JAK2 mutations to be favorable (Blood 2010). Further, in some studies, mutations known to affect disease progression in MF pre-HCT (high-molecular risk or HMR mutations) have not been predictors of post-HCT outcomes. Here we present outcomes in all MF patients undergoing HCT at Dana-Farber Cancer Institute (DFCI) from 2000-2020 and examine clinical and genetic predictors of outcomes, stratified by year of HCT (<2011 or pre-2011 and >2011 or post-2011). Of note, ruxolitinib was available for use post-2011 only, following its FDA approval that year. Methods: Patients with MF who underwent HCT from 2000-2020 at DFCI (n=166) were included in this retrospective cohort analysis. Outcomes were analyzed for the whole cohort as well as sub-group analyses in the pre-2011 (n=39) and post-2011 era (n=127). Mutational analysis was performed by targeted next-generation sequencing of available pre-HCT samples in 115/127 patients in the post-2011 era. Results: Median age was 61 years and follow up 56.2 months. 25% had massive splenomegaly. DIPSS-plus scores were high in 45.2% and 45.1% received rux. HCT and patient characteristics (including KPS and median time from diagnosis to HCT) were similar pre and post 2011, with differences in conditioning regimens. Majority (77.1%) had HLA-matched donors, underwent reduced intensity (69.3%) HCT with peripheral blood stem-cells (92.2%) and received tacrolimus/methotrexate based GVHD prophylaxis (90.1%). FluMel100/140 (n=65), FluBu4 (n=34) and FluBu2 (n=11) were commonly used in the post-2011 era, while Flu/Bu1 (n= 34), ablative Bu/Cy (n=5) and Cy/TBI (n=12) were predominant pre-2011. Few patients underwent pre-HCT splenectomy (10.2%) or splenic irradiation (9%). Median D100 donor chimerism was 68% for Flu/Bu1, 86% for Flu/Bu2, 98% for Flu/Bu4, and 100% for Flu/Mel100-140. At 4 years, overall survival (OS) for the whole cohort was 58% (50,66), progression-free survival (PFS) 55% (47,63), non-relapse mortality (NRM) 23% (17,30) and relapse rate 21%. 4 year OS was significantly better in the post-2011 era compared to pre-2011 (69% vs 26%, p<0.0001), as was PFS, driven by lower relapse rates in the post-2011 era (15% vs 41%, p<0.0001). 4-yr NRM was also marginally better (20% vs 33%, p= 0.17). In the post-2011 era, 115/127 patients had NGS panel-based genetic analysis prior to HCT (Fig 1), demonstrating 91.3% of patients had at least one driver mutation (JAK2 62.6%, CAL-R 13.9%, MPL 14.8%) while 8.7% patients were triple negative. The most frequent cooccurring mutations were ASXL1 (40%), TET2 (28.7%) and U2AF1 (17.4%).In univariable analysis (UVA), the presence of JAK2 mutations (JAK2+) was associated with superior PFS (HR 0.5, 95% CI 0.27,0.94, p= 0.03) while CALR (HR 1.86, p=0.12) and MPL (HR:1.21, p=0.65) mutations were not significant for PFS. Triple negative patients showed a trend towards poorer PFS (HR 2.27, p=0.05). OS results mirrored those of PFS. No cooccurring myeloid gene mutations were associated with PFS or OS. Superior 4-year OS for JAK2+ versus JAK2- recipients (80% versus 58%, p=0.01) is shown in Figure 2. TP53 (p=0.03), TET2 (p=0.015) and EZH2 (p=0.002) mutations were associated with higher relapse rates in UVA. Patients with JAK2+ and cooccurring ASXL1 mutations had poorer OS compared to those with JAK2+/no cooccurring ASXL1 mutations (HR=2.69, p=0.031). Combining clinical and molecular features in the post-2011 era, in MVA, only DIPSS high (HR=4.76, p=0.03) and KPS < 90% (HR=2.66, p=0.024) were significantly associated with poorer OS/PFS while JAK2+ (HR=0.32, 95% CI 0.14,0.75,p=0.009) was significantly associated with better OS/PFS; JAK inhibitor use spleen size/management, conditioning regimen or other mutations including HMR mutations and TP53 were not significantly associated with OS/PFS. Conclusions: In this single-institution retrospective analysis, the presence of a JAK2-V617F mutation was associated with better OS and PFS following HCT in MF in the post-2011 era. Survival outcomes are significantly better in the post-2011 era compared to pre-2011, in our cohort.

Background: Despite tremendous progress in the treatment of myelofibrosis in the last decade, the only curative therapy remains allogeneic hematopoietic cell transplantation (HCT). There are now three FDA-approved JAK inhibitors (JAKi) to treat symptomatic splenomegaly and constitutional symptoms in myelofibrosis, although these agents do not prevent disease progression. Discontinuation of JAKi before (pre) HCT is challenging as patients can experience return of symptoms. Thus, ruxolitinib is often continued during HCT in an off-label fashion; however, little is known about the safety and efficacy of this approach. Furthermore, there is increasing use of ruxolitinib after (post) HCT, following its approval in treating refractory graft-versus-host disease (GVHD). Methods: We previously reported the interim results of a phase II, multicenter, investigator-initiated trial investigating ruxolitinib given pre-, during- and for 1-year post-HCT for patients with primary or secondary MF (NCT03427866). Here we present the results of the study with full accrual of 43 patients. The primary endpoint was 1-year GVHD-free, relapse-free survival (GRFS). Secondary endpoints included overall (OS) and progression free survival (PFS), engraftment, and incidence of acute and chronic GVHD. Patients were treated with reduced intensity conditioning with fludarabine (30mg/m 2/d x 5 days) and melphalan (100 or 140mg/m 2 x 1). Patients received peripheral blood stem cell grafts from either 7/8 or 8/8 HLA-matched donors. GVHD prophylaxis consisted of standard tacrolimus and methotrexate. Results: 44 patients with myelofibrosis were enrolled and 43 underwent HCT between 9/2018 and 5/2023. Median age was 66 (range 46-75), 37% were female (n=16), and most patients had DIPSS intermediate-2/high risk (n=36), 60% (n=27) were receiving ruxolitinib prior to enrolling on the study. Most (n=35) received 8/8 matched unrelated transplants and had measurable splenomegaly by either ultrasound or CT (n=40) prior to HCT. At baseline, 25 patients had JAK2 mutation, 8 had CALR and 5 had MPL mutations, 13 patients had ASXL1 mutations (Figure 1). No differences in outcomes were observed by pre-HCT mutational status. At day 100, all but one patient had undetectable mutations by next generation sequencing. The most common grade 3/4 hematologic adverse events (AE) were anemia (n=12), thrombocytopenia (14), neutropenia (n=11). Non-hematologic grade 3/4 events included hypertriglyceridemia (n=3). Median time to neutrophil engraftment was 15 days (range 4-38), one patient did not engraft. Median time to platelet (>20x10 9) engraftment was 25 days (range 11-145). At day 30, 2 patients had a donor-cell chimerism below 90% (one ultimately relapsed) and at day 100, 2 patients had chimerism below 90 (one ultimately relapsed). With a median follow up among survivors of 13 months (range 0.7 - 26), 1-year GRFS was 74%. OS, PFS and cumulative incidence of NRM and disease relapse were, 86%, 79%, 10%, and 10%, respectively (Figure 2). The cumulative incidence of grade III-IV acute GVHD at 6 months was 2.4%. Only one patient had grade IV acute GVHD. One year moderate/severe chronic GVHD was 11%In univariable analysis, thrombocytopenia of <40 x 10 9/L pre-HCT was associated with worse OS (1-yr OS: 64% vs 92%, p=0.008). Splenomegaly (defined as >17 cm) was associated with worse PFS (1-yr PFS: 100% vs 64%, p=0.046). There was an improvement in OS in patients that received ruxolitinib and achieved response pre-HCT compared to those that did not but this did not achieve statistical significance (1-yr OS 100% vs 74%, p=0.054). Discussion: This study is the first, multicenter trial to test ruxolitinib use pre-, during- and post-HCT in patients with myelofibrosis. Our results demonstrate safety and feasibility of this approach. Favorable rates of engraftment were observed with ongoing ruxolitinib administration even through hematopoietic nadir. We continue to demonstrate favorable outcomes of PFS, OS and GRFS with a low incidence of severe acute and chronic GVHD. Our results strongly support the use of ruxolitinib in the peri- and post-transplant setting for patients with myelofibrosis undergoing HCT.