Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-ß Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA-sequencing revealed that CALRdel52 led to the expansion of TGF-ß1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGF-ß antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, while T cell depletion abrogated the protective effects conferred by neutralizing TGF-ß. TGF-ß1 reduced perforin and TNF-α production by T cells in vitro. TGF-ß1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGF-ß1 expression. In four independent patient cohorts, TGF-ß1 expression was increased in the BM of MPN patients compared to healthy individuals, and the BM of MPN patients contained a higher frequency of Treg compared to healthy individuals. Together, this study identified an ERK/Sp1/TGF-ß1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity.

Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) encompass a diverse group of hematologic disorders driven by mutations in JAK2, CALR, or MPL. The prevailing working model explaining how these driver mutations induce different disease phenotypes is based on the decisive influence of the cellular microenvironment and the acquisition of additional mutations. Here, we report increased levels of chromatin segregation errors in hematopoietic cells stably expressing CALRdel52 or JAK2V617F mutations. Our investigations employing murine 32DMPL and human erythroleukemic TF-1MPL cells demonstrate a link between CALRdel52 or JAK2V617F expression and a compromised spindle assembly checkpoint (SAC), a phenomenon contributing to error-prone mitosis. This defective SAC is associated with imbalances in the recruitment of SAC factors to mitotic kinetochores upon CALRdel52 or JAK2V617F expression. We show that JAK2 mutant CD34 + MPN patient-derived cells exhibit reduced expression of the master mitotic regulators PLK1, aurora kinase B, and PP2A catalytic subunit. Furthermore, the expression profile of mitotic regulators in CD34 + patient-derived cells allows to faithfully distinguish patients from healthy controls, as well as to differentiate primary and secondary myelofibrosis from essential thrombocythemia and polycythemia vera. Altogether, our data suggest alterations in mitotic regulation as a potential driver in the pathogenesis in MPN.

Engineering Mesoscopic 3D Tumor Models with a Self-Organizing Vascularized Matrix.

Advanced in vitro systems such as multicellular spheroids and lab-on-a-chip devices have been developed, but often fall short in reproducing the tissue scale and self-organization of human diseases. A bioprinted artificial tumor model is introduced with endothelial and stromal cells self-organizing into perfusable and functional vascular structures. This model uses 3D hydrogel matrices to embed multicellular tumor spheroids, allowing them to grow to mesoscopic scales and to interact with endothelial cells. It is shown that angiogenic multicellular tumor spheroids promote the growth of a vascular network, which in turn further enhances the growth of cocultivated tumor spheroids. The self-developed vascular structure infiltrates the tumor spheroids, forms functional connections with the bioprinted endothelium, and can be perfused by erythrocytes and polystyrene microspheres. Moreover, cancer cells migrate spontaneously from the tumor spheroid through the self-assembled vascular network into the fluid flow. Additionally, tumor type specific characteristics of desmoplasia, angiogenesis, and metastatic propensity are preserved between patient-derived samples and tumors derived from this same material growing in the bioreactors. Overall, this modular approach opens up new avenues for studying tumor pathophysiology and cellular interactions in vitro, providing a platform for advanced drug testing while reducing the need for in vivo experimentation.

Exploiting Synthetic Lethality between Germline BRCA1 Haploinsufficiency and PARP Inhibition in JAK2V617F-Positive Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are rare hematologic disorders characterized by clonal hematopoiesis. Familial clustering is observed in a subset of cases, with a notable proportion exhibiting heterozygous germline mutations in DNA double-strand break repair genes (e.g., BRCA1). We investigated the therapeutic potential of targeting BRCA1 haploinsufficiency alongside the JAK2V617F driver mutation. We assessed the efficacy of combining the PARP inhibitor olaparib with interferon-alpha (IFNα) in CRISPR/Cas9-engineered Brca1+/- Jak2V617F-positive 32D cells. Olaparib treatment induced a higher number of DNA double-strand breaks, as demonstrated by γH2AX analysis through Western blot (p = 0.024), flow cytometry (p = 0.013), and confocal microscopy (p = 0.071). RAD51 foci formation was impaired in Brca1+/- cells compared to Brca1+/+ cells, indicating impaired homologous recombination repair due to Brca1 haploinsufficiency. Importantly, olaparib enhanced apoptosis while diminishing cell proliferation and viability in Brca1+/- cells compared to Brca1+/+ cells. These effects were further potentiated by IFNα. Olaparib induced interferon-stimulated genes and increased endogenous production of IFNα in Brca1+/- cells. These responses were abrogated by STING inhibition. In conclusion, our findings suggest that the combination of olaparib and IFNα presents a promising therapeutic strategy for MPN patients by exploiting the synthetic lethality between germline BRCA1 mutations and the JAK2V617F MPN driver mutation.

Clonogenic assays improve determination of variant allele frequency of driver mutations in myeloproliferative neoplasms.

Molecular diagnostics moves more into focus as technology advances. In patients with myeloproliferative neoplasms (MPN), identification and monitoring of the driver mutations have become an integral part of diagnosis and monitoring of the disease. In some patients, none of the known driver mutations (JAK2V617F, CALR, MPL) is found, and they are termed "triple negative" (TN). Also, whole-blood variant allele frequency (VAF) of driver mutations may not adequately reflect the VAF in the stem cells driving the disease. We reasoned that colony forming unit (CFU) assay-derived clonogenic cells may be better suited than next-generation sequencing (NGS) of whole blood to detect driver mutations in TN patients and to provide a VAF of disease-driving cells. We have included 59 patients carrying the most common driver mutations in the establishment or our model. Interestingly, cloning efficiency correlated with whole blood VAF (p = 0.0048), suggesting that the number of disease-driving cells correlated with VAF. Furthermore, the clonogenic VAF correlated significantly with the NGS VAF (p < 0.0001). This correlation was lost in patients with an NGS VAF <15%. Further analysis showed that in patients with a VAF <15% by NGS, clonogenic VAF was higher than NGS VAF (p = 0.003), suggesting an enrichment of low numbers of disease-driving cells in CFU assays. However, our approach did not enhance the identification of driver mutations in 5 TN patients. A significant correlation of lactate dehydrogenase (LDH) serum levels with both CFU- and NGS-derived VAF was found. Our results demonstrate that enrichment for clonogenic cells can improve the detection of MPN driver mutations in patients with low VAF and that LDH levels correlate with VAF.

Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. SIGNIFICANCE: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

Progression of Myeloproliferative Neoplasms (MPN): Diagnostic and Therapeutic Perspectives.

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematologic malignancies, including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), as well as post-PV-MF and post-ET-MF. Progression to more symptomatic disease, such as overt MF or acute leukemia, represents one of the major causes of morbidity and mortality. There are clinically evident but also subclinical types of MPN progression. Clinically evident progression includes evolution from ET to PV, ET to post-ET-MF, PV to post-PV-MF, or pre-PMF to overt PMF, and transformation of any of these subtypes to myelodysplastic neoplasms or acute leukemia. Thrombosis, major hemorrhage, severe infections, or increasing symptom burden (e.g., pruritus, night sweats) may herald progression. Subclinical types of progression may include increases in the extent of bone marrow fibrosis, increases of driver gene mutational allele burden, and clonal evolution. The underlying causes of MPN progression are diverse and can be attributed to genetic alterations and chronic inflammation. Particularly, bystander mutations in genes encoding epigenetic regulators or splicing factors were associated with progression. Finally, comorbidities such as systemic inflammation, cardiovascular diseases, and organ fibrosis may augment the risk of progression. The aim of this review was to discuss types and mechanisms of MPN progression and how their knowledge might improve risk stratification and therapeutic intervention. In view of these aspects, we discuss the potential benefits of early diagnosis using molecular and functional imaging and exploitable therapeutic strategies that may prevent progression, but also highlight current challenges and methodological pitfalls.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes.

Calreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

Early and late stage MPN patients show distinct gene expression profiles in CD34+ cells.

Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF), are hematological disorders of the myeloid lineage characterized by hyperproliferation of mature blood cells. The prediction of the clinical course and progression remains difficult and new therapeutic modalities are required. We conducted a CD34+ gene expression study to identify signatures and potential biomarkers in the different MPN subtypes with the aim to improve treatment and prevent the transformation from the rather benign chronic state to a more malignant aggressive state. We report here on a systematic gene expression analysis (GEA) of CD34+ peripheral blood or bone marrow cells derived from 30 patients with MPN including all subtypes (ET (n = 6), PV (n = 11), PMF (n = 9), secondary MF (SMF; post-ET-/post-PV-MF; n = 4)) and six healthy donors. GEA revealed a variety of differentially regulated genes in the different MPN subtypes vs. controls, with a higher number in PMF/SMF (200/272 genes) than in ET/PV (132/121). PROGENγ analysis revealed significant induction of TNFα/NF-κB signaling (particularly in SMF) and reduction of estrogen signaling (PMF and SMF). Consistently, inflammatory GO terms were enriched in PMF/SMF, whereas RNA splicing-associated biological processes were downregulated in PMF. Differentially regulated genes that might be utilized as diagnostic/prognostic markers were identified, such as AREG, CYBB, DNTT, TIMD4, VCAM1, and S100 family members (S100A4/8/9/10/12). Additionally, 98 genes (including CLEC1B, CMTM5, CXCL8, DACH1, and RADX) were deregulated solely in SMF and may be used to predict progression from early to late stage MPN.

The Unfolded Protein Response Is a Major Driver of LCN2 Expression in BCR-ABL- and JAK2V617F-Positive MPN.

Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1-JNK-NF-κB-C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Differential roles of STAT1 and STAT2 in the sensitivity of JAK2V617F- vs. BCR-ABL-positive cells to interferon alpha.

BACKGROUND: Interferon alpha (IFNa) monotherapy is recommended as the standard therapy in polycythemia vera (PV) but not in chronic myeloid leukemia (CML). Here, we investigated the mechanisms of IFNa efficacy in JAK2V617F- vs. BCR-ABL-positive cells. METHODS: Gene expression microarrays and RT-qPCR of PV vs. CML patient PBMCs and CD34+ cells and of the murine cell line 32D expressing JAK2V617F or BCR-ABL were used to analyze and compare interferon-stimulated gene (ISG) expression. Furthermore, using CRISPR/Cas9n technology, targeted disruption of STAT1 or STAT2, respectively, was performed in 32D-BCR-ABL and 32D-JAK2V617F cells to evaluate the role of these transcription factors for IFNa efficacy. The knockout cell lines were reconstituted with STAT1, STAT2, STAT1Y701F, or STAT2Y689F to analyze the importance of wild-type and phosphomutant STATs for the IFNa response. ChIP-seq and ChIP were performed to correlate histone marks with ISG expression. RESULTS: Microarray analysis and RT-qPCR revealed significant upregulation of ISGs in 32D-JAK2V617F but downregulation in 32D-BCR-ABL cells, and these effects were reversed by tyrosine kinase inhibitor (TKI) treatment. Similar expression patterns were confirmed in human cell lines, primary PV and CML patient PBMCs and CD34+ cells, demonstrating that these effects are operational in patients. IFNa treatment increased Stat1, Stat2, and Irf9 mRNA as well as pY-STAT1 in all cell lines; however, viability was specifically decreased in 32D-JAK2V617F. STAT1 or STAT2 knockout and reconstitution with wild-type or phospho-deficient STAT mutants demonstrated the necessity of STAT2 for IFNa-induced STAT1 phosphorylation in BCR-ABL- but not in JAK2V617F-expressing cells. STAT1 was essential for IFNa activity in both BCR-ABL- and JAK2V617F-positive cells. Furthermore, ChIP experiments demonstrate higher repressive and lower active chromatin marks at the promoters of ISGs in BCR-ABL-expressing cells. CONCLUSIONS: JAK2V617F but not BCR-ABL sensitizes MPN cells to interferon, and this effect was dependent on STAT1. Moreover, STAT2 is a survival factor in BCR-ABL- and JAK2V617F-positive cells but an IFNa-sensitizing factor solely in 32D-JAK2V617F cells by upregulation of STAT1 expression.

JAK2V617F but not CALR mutations confer increased molecular responses to interferon-α via JAK1/STAT1 activation.

Pegylated interferon-α (peg-IFNa) treatment induces molecular responses (MR) in patients with myeloproliferative neoplasms (MPNs), including partial MR (PMR) in 30-40% of patients. Here, we compared the efficacy of IFNa treatment in JAK2V617F- vs. calreticulin (CALR)-mutated cells and investigated the mechanisms of differential response. Retrospective analysis of MPN patients treated with peg-IFNa demonstrated that patients harboring the JAK2V617F mutation were more likely to achieve PMR than those with mutated CALR (p = 0.004), while there was no significant difference in hematological response. In vitro experiments confirmed an upregulation of IFN-stimulated genes in JAK2V617F-positive 32D cells as well as patient samples (peripheral blood mononuclear cells and CD34+ hematopoietic stem cells) compared to their CALR-mutated counterparts, and higher IFNa doses were needed to achieve the same IFNa response in CALR- as in JAK2V617F-mutant 32D cells. Additionally, Janus-activated kinase-1 (JAK1) and signal transducers and activators of transcription 1 (STAT1) showed constitutive phosphorylation in JAK2V617F-mutated but not CALR-mutated cells, indicating priming towards an IFNa response. Moreover, IFN-induced growth arrest was counteracted by selective JAK1 inhibition but enhanced by JAK2 inhibition. In conclusion, our data suggest that, clinically, higher doses of IFNa are needed in CALR-mutated vs. JAK2V617F-positive patients and we suggest a model of JAK2V617F-JAK1/STAT1 crosstalk leading to a priming of JAK2V617F-positive cells to IFNa resulting in differential sensitivity.

Nrf2 Is a Central Regulator of Metabolic Reprogramming of Myeloid-Derived Suppressor Cells in Steady State and Sepsis.

Arising in inflammatory conditions, myeloid-derived suppressor cells (MDSCs) are constantly confronted with intracellular and extracellular reactive oxygen species molecules and oxidative stress. Generating mice with a constitutive activation of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) we show a pivotal role of the antioxidant stress defense for development of these immune-modulatory cells. These mice are characterized by a massive increase of splenic CD11b+Gr-1+ cells, which exhibit typical suppressive characteristics of MDSCs. Whole transcriptome analysis revealed Nrf2-dependent activation of cell cycle and metabolic pathways, which resemble pathways in CD11b+Gr-1+ MDSCs expanded by in vivo LPS exposure. Constitutive Nrf2 activation thereby regulates activation and balance between glycolysis and mitochondrial metabolism and hence expansion of highly suppressive MDSCs, which mediate protection in LPS-induced sepsis. Our study establishes Nrf2 as key regulator of MDSCs and acquired tolerance against LPS-induced sepsis.