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Primary Myelofibrosis (PMF) is an aggressive myeloproliferative neoplasm (MPN) characterized by constitutional symptoms, cytopenias, splenomegaly, extramedullary hematopoiesis, bone marrow (BM) fibrosis, and a propensity to transform to acute myeloid leukemia (AML). Despite advances in understanding the underlying genetic abnormalities in MPN and the development of JAK2 inhibitors to treat MF, there is an urgent need to devise new treatment strategies, particularly for triple negative myelofibrosis (TN-MF) cases that lack mutations in the JAK2 kinase pathway and who have inferior outcomes. To identify potential oncogenic drivers in TN-MF, we performed cytogenetic analyses and targeted exome sequencing of 98 genes commonly mutated in myeloid malignancies in 584 MF patients identified from the Total Cancer Care database at Moffitt Cancer Center. Notably, there were no significant differences in the somatic mutation profiles (other than JAK2 activating mutations) between TN vs. JAK2/CALR/MPL mutant MF patients. However, trisomy of chromosome 8 occurs more frequently in TN-MF vs. other subtypes (26.7% vs. 6.2%, p=0.0001). To identify oncogenic drivers in trisomy 8+ TN-MF, we performed scRNA-seq analysis from 18,651 cells ( n=15,850 from 3 normal donors and n=2,801 from a trisomy 8+ TN-MF patient) and identified a total of 1,260 genes that are differentially regulated in hematopoietic stem cells (HSCs) of trisomy 8+ TN-MF compared to normal donors. Among these, 131 genes were located on chromosome 8 and MYC was one of the top 5 genes that are significantly upregulated in trisomy 8+ HSCs. Additional immunohistochemistry staining demonstrated that MYC protein levels in BM cells were significantly higher in trisomy 8+ TN-MF patients vs. trisomy 8- patients. To assess whether MYC might be a driver of MPN, we established an Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC transgenic mouse model that inducibly overexpresses MYC in HSCs following treatment of polyinosinic-polycytidylic acid (pIpC). Forced MYC expression induced profound anemia, monocytosis, megakaryocytic atypia, splenomegaly, increased BM collagen deposition/fibrosis, extramedullary hematopoiesis in spleen and liver, and significantly reduced OS vs. Mx1-Cre +/- wild type controls (median OS 258 days vs. not reached (NR), p<0.0001). MYC also promoted expansion of HSCs, myeloid progenitors, and Gr-1 +/CD11b + mature myeloid cells in BM and spleen; thus, MYC was sufficient to provoke MF-like disease independent of JAK2 pathway mutations. Subsequent scRNA-seq analysis of 25,232 cells ( n=13,552 from control and n=11,680 from a MYC transgenic mouse) revealed that S100a9 mRNA levels were increased by MYC in most major hematopoietic cell types. Supporting oncogenic roles of S100A9 in MYC-driven MF, deletion of S100a9 in Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC;S100a9 -/- mice abolished MYC-induced MF phenotypes and significantly improved OS (median OS NR vs. 225 days, p=0.0345). To test whether inhibition of MYC can suppress MF disease progression in vivo, lethally irradiated CD45.1 +/CD45.2 + WT mice were transplanted with BM cells harvested from CD45.2 + Mx1-Cre +/-;Rosa26 LSL-MYC/LSL-MYC mice that were treated with pIpC 20 weeks prior to transplant. MYCi975, a small molecule that inhibits MYC, reduced MYC and S100a9 protein levels in BM and spleen cells. Further, MYCi975 effectively suppressed MYC-driven MF phenotypes and disease progression, and significantly improved OS (median OS NR vs. 252 days, p=0.0109). In summary, our studies are the first to describe an oncogenic role of MYC in MF pathogenesis, where MYC provokes an alarmin-driven inflammatory circuit, and where the MYC-S100A9 axis represents a therapeutic vulnerability for TN-MF patients. Accordingly, our results provide a strong rationale for testing agents targeting MYC or S100A9 in early phase clinical trials in MPNs having increased MYC levels or activity.

Introduction: Sickle cell disease (SCD) is the most common inherited hemoglobinopathy affecting populations across the United States. Several well-established patient-centered comprehensive care models have been in practice for preventive care and pain management. However, access to hematopoietic stem cell transplant (HSCT) and clinical trials for sickle cell gene therapies are limited, especially in pediatric and adult centers with limited HSCT-related resources. We describe a patient-centered collaborative model in the Upper Midwest United States to improve patient education and engagement as well as access to HSCT and gene therapy trials for patients with SCD. Collaborative Care Model: Since August 2021, centers across the Midwest meet monthly to improve access for HSCT and gene therapy as well as education about these therapies for individuals with SCD. The teams include: hematology and transplant teams from University of Minnesota, Comprehensive Sickle Cell Care team from Children's Hospital of Minneapolis, hematology and transplant team from Mayo Children's Hospital, hematology team from Essential Health, Duluth, Minnesota, hematology team from Sanford Children's Hospital Fargo and Sioux Falls and hematology team from Blank Children's Hospital, Des Moines, Iowa. This collaborative approach involves several stakeholders including an SCD community advocacy organization, hematology care providers across several rural as well as community/university hospitals, social workers, nurse coordinators, HSCT/gene therapy providers, as well as representatives from National Marrow Donor Program. Individual patient needs and available community support as well as institutional resources are discussed at length during monthly virtual meetings and a pre-intervention plan is established to ensure smooth transition of care to and from primary centers (Figure 1). Ongoing communication with the primary team helps with efficient transition of care after transplant as patients continue ongoing monitoring and care closer to home. Community partners help with early education of the families and provide necessary resources and support. Institutional partners like NMDP provide support with immune typing and discuss ongoing support and other resources available for providers and families. Results/Impact: Since the implementation of this collaborative model, there has been a steady increase in early referrals for education regarding HSCT and potential gene therapies trials for patients with SCD. There has been about 100% increase in new patient referrals since the implementation of this collaborative model compared to the average new referrals in the previous years (2021-22 vs 2018-19; Figure 2). Enrollment in HSCT as well as gene therapy clinical trials also has increased 2-3 folds since the implementation of the collaborative care model. It has helped established early discussion and longitudinal care and has also led to ongoing evaluation and discussions for availability of optimal donors, determining eligibility for HSCT and gene therapy trials as well as providing ongoing support and education to the sickle cell community and providers regarding the available therapies for SCD. This has also facilitated the transplant team establish earlier and better rapport with patients and their families and provide necessary education with an in-depth discussion regarding the potential benefits and risks of therapies such as HSCT and gene therapy. These discussions often occur over multiple visits and helps address psychological as well social barriers with a prospective evaluation. Additionally, the ongoing discussion among the providers helps with longitudinal monitoring of the patients and provide an assessment of the risks and benefits of therapies as the patient's disease course continues to evolve. This multi-stake holder collaborative approach continues to provide a well-established platform for discussion of newer therapies and access to open clinical trials, available resources for patients and families, and improve patient care and experience. The impact of this collaboration is noted in significant increase in early referrals and educational consults for HSCT and gene therapies, improved patient engagement as well as the acceptability and participation in clinical trials.

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question. To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp‘) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts ( p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status. Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3) Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mut TP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block. Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive toDYRK1A inhibitors, while healthy control CD34+ cells were unaffected Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in γ-H2AX foci after etoposide treatment or irradiation ( p<0.01 for both). A second mechanism of leukemogenesis emerged from geneset enrichment analyses, which suggested enhanced JAK-STAT signaling in chr21amp BPMPN cells and downregulation in the CRISPR DYRK1A KO context. We validated this by showing that DYRK1A overexpression activates and potentiates STAT5B transcriptional activity in a luciferase reporter assay. Finally, we noted that the STAT target BCL2 was selectively upregulated in chr21amp cells. BCL2 inhibition showed strong synergy with DYRK1A inhibitors for induction of BPMPN cell apoptosis (Bliss synergy score 15). Collectively, these findings define the chr21amp event as a novel prognostic biomarker in BPMPN. We pinpoint DYRK1A amplification as a central driver of genomic instability and exacerbated JAK-STAT signalling, for the first time linking chromothripsis to a specific druggable target ( Fig 1B).

Abstract During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3 , Chchd6 , and Mitofilin . We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.