Proteogenetic drug response profiling elucidates targetable vulnerabilities of myelofibrosis.

Myelofibrosis is a hematopoietic stem cell disorder belonging to the myeloproliferative neoplasms. Myelofibrosis patients frequently carry driver mutations in either JAK2 or Calreticulin (CALR) and have limited therapeutic options. Here, we integrate ex vivo drug response and proteotype analyses across myelofibrosis patient cohorts to discover targetable vulnerabilities and associated therapeutic strategies. Drug sensitivities of mutated and progenitor cells were measured in patient blood using high-content imaging and single-cell deep learning-based analyses. Integration with matched molecular profiling revealed three targetable vulnerabilities. First, CALR mutations drive BET and HDAC inhibitor sensitivity, particularly in the absence of high Ras pathway protein levels. Second, an MCM complex-high proliferative signature corresponds to advanced disease and sensitivity to drugs targeting pro-survival signaling and DNA replication. Third, homozygous CALR mutations result in high endoplasmic reticulum (ER) stress, responding to ER stressors and unfolded protein response inhibition. Overall, our integrated analyses provide a molecularly motivated roadmap for individualized myelofibrosis patient treatment.

Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model.

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic ("don't eat me") CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6Chi monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more "pro-phagocytic," suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages.

Calreticulin mutations affect its chaperone function and perturb the glycoproteome.

Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.

Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma.

Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Enhanced engraftment of human myelofibrosis stem and progenitor cells in MISTRG mice.

The engraftment potential of myeloproliferative neoplasms in immunodeficient mice is low. We hypothesized that the physiological expression of human cytokines (macrophage colony-stimulating factor, interleukin-3, granulocyte-macrophage colony-stimulating factor, and thrombopoietin) combined with human signal regulatory protein α expression in Rag2-/-Il2rγ-/- (MISTRG) mice might provide a supportive microenvironment for the development and maintenance of hematopoietic stem and progenitor cells (HSPC) from patients with primary, post-polycythemia or post-essential thrombocythemia myelofibrosis (MF). We show that MISTRG mice, in contrast to standard immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ and Rag2-/-Il2rγ-/- mice, supported engraftment of all patient samples investigated independent of MF disease stage or risk category. Moreover, MISTRG mice exhibited significantly higher human MF engraftment levels in the bone marrow, peripheral blood, and spleen and supported secondary repopulation. Bone marrow fibrosis development was limited to 3 of 14 patient samples investigated in MISTRG mice. Disease-driving mutations were identified in all xenografts, and targeted sequencing revealed maintenance of the primary patient sample clonal composition in 7 of 8 cases. Treatment of engrafted mice with the current standard-of-care Janus kinase inhibitor ruxolitinib led to a reduction in human chimerism. In conclusion, the established MF patient-derived xenograft model supports robust engraftment of MF HSPCs and maintains the genetic complexity observed in patients. The model is suited for further testing of novel therapeutic agents to expedite their transition into clinical trials.