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Annals of hematology 102(2), 349-358 (2022). doi:10.1007/s00277-022-05080-7, Published by Springer, Berlin ; Heidelberg ; New York

Additional file 1. Supplemental methods, supplemental figures, and supplemental tables S1, S2, and S4.

We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNF��/NF��B signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-�� further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-�� provides a possible mechanism of how interferon-�� preferentially targets the JAK2 mutant clone.

Figure S6. Confirmation of successful STAT1 or STAT2 knockout. Western blotting of several 32D-BCR-ABL or 32D-JAK2V617F STAT1 or STAT2 knockout clones. STAT2 antibody was used to confirm the knockout, and GAPDH served as the loading control. 32D cells were WEHI starved for 24 h before starting the experiment. wt – wild-type clones, ko – knockout clones, het – presumed heterozygous clones (PDF 134 kb)

Supplementary Material & Methods. Table S1. Primer for cloning STAT1 and STAT2 with included restriction sites. Table S2. STAT1 and STAT2 primers for mutagenesis. Table S3. RT-qPCR primer for gene expression analysis. Table S4. Western blot antibodies. Table S5. gRNA Oligonucleotides. Table S6. ChIP antibodies. Table S7. ChIP-PCR Primer. Table S8. GSEA of a set of 45 interferon-stimulated genes (ISG). Table S9. Statistics for the enrichment analysis of transcription factor binding sites (TFBS) in acetylation (H3K9ac) peaks. (DOCX 44 kb)

Figure S9. Full RT-qPCR panels of tested ISGs. Illustration of the RT-qPCR results of 32D-BCR-ABL- and 32D-JAK2V617F-WT or -STATko or -STAT1(Y/F) and STAT2(Y/F) reconstituted cell clones treated with IFNa (100 U/ml) or left untreated (triplicate), corresponding to the data given in Figs. 3f and 4d. (a) Stat1 and Stat2, (b) Irf7 and Irf9. Stat2 qPCR primer detected the ectopically expressed Stat2 mRNA, explaining the strong upregulation, and endogenous Stat2 can thus not be evaluated in the reconstituted experiments (gray bars). Independent experiments were performed three times. (PDF 56 kb)

Figure S8. Illustration of pY-STAT1 in 32D-JAK2V617F cells in the absence of IFNa. STAT1 and pY-STAT1 stainings of Fig. 4c, shown with increased contrast. The arrows are indicating the pY-STAT1 bands in 32D-BCR-ABL- and 32D-JAK2V617F-STATko cells. (PDF 203 kb)

Figure S7. Comparison of CRISPR/Cas9 manipulated 32D cell lines treated with 100 U IFNa in survival and titration of lower IFNa dosages. Indicated (A) 32D-BCR-ABL and (B) 32D-JAK2V617F cell lines were analyzed in an MTT assay and treated with 100 U IFNa for 72 h (abstracted from Fig. 4a, b). Absorption was normalized to untreated control cells and statistically analyzed using a t test. Mean values ± SD are indicated. *p

Figure S2. MTT assay of 32D-BCR-ABL and 32D-JAK2V617F cells treated with IFNa. 32D-BCR-ABL-(blue) and 32D-JAK2V617F-(red) positive cells were treated with IFNa (0–104 U/ml) alone (continuous lines) or in combination with 0.1 μM imatinib (IM) or ruxolitinib (Rux) (dotted lines) for 72 h and the viability was measured by MTT. Viability was normalized to the untreated control and mean values ± SD are depicted. The respective 32D cells were WEHI starved for 24 h before starting the experiments. Experiments were performed in triplicate and conducted three times. (PDF 27 kb)

Figure S1. Knockout of STAT1 or STAT2 with CRISPR/Cas9n technology. Four guide RNAs have been generated for STAT1 or STAT2 knockout in 32D-BCR-ABL and 32D-JAK2V617F cells, respectively. Excised exons are given. (PDF 19 kb)

Figure S4. Effect of extrinsic soluble factors on gene expression in 32D-EV- or 32D-JAK2V617F-positive cells. Supernatant of WEHI-starved 32D-EV- or 32D-JAK2V617F-positive cells was generated overnight, and after removal of the cells, fresh EV (green) or JAK2V617F-(red) positive cells were incubated with the supernatant for 2 h prior to RNA extraction to analyze the expression of IFN target genes. Mean ± SD values are shown as % of Gapdh. Independent experiments were performed three times and in triplicate, respectively. (PDF 25 kb)

Figure S3. BCR-ABL reduces ISG expression in 32D cells. Gene expression microarray analysis of 32D-EV, 32D-BCR-ABL, or 32D-JAK2V617F cells. Fold change of gene expression is shown, depicting downregulation of the analyzed gene in blue and upregulation in red. (PDF 134 kb)

Figure S5. Correlation of ISG expression and JAK2V617F allelic burden and Western blot of 32D EV, BCR-ABL, or JAK2V617F cells. A, ISG expression (% of GAPDH) and JAK2V617F allelic burden (in %) were plotted against each other and a significant correlation was only found for IRF9 (p = 0.0492). Samples with additional mutations (TET2, ASXL1, or EZH2) were highlighted with a red circle. B, Western blot analysis of pY-STAT1 and overall STAT1 protein after TKI and/or IFNa treatment (4 h) of 32D EV (only −/+ IFNa), BCR-ABL, or JAK2V617F cells. All cell lines were starved of WEHI (source of IL-3) for 24 h before treatment. GAPDH served as the loading control. The same Western blot is shown in Fig. 2c lacking 32D EV cells. (PDF 74 kb)

Figure S10. H3K9 acetylation profile at ISGs. The ChIP-seq acetylation was visualized with the Integrative Genomics Viewer (IGV). Shown are the H3K9 acetylation peaks at the genomic regions of Irf1, Irf17, Irf9, and Stat1 in 32D-JAK2V7F (JAK2V617F) (red), 32D-BCR-ABL (blue), and 32D-EV (green). (PDF 108 kb)

Supplementary methods. Table S1. Bone marrow fibrosis grade. Table S2. Treatment-emergent AEs of any grade that occurred in ≥ 2 patients. Table S3. AEs leading to treatment interruption or modification. Table S4. Mean (± SD) total and unbound steady-state vismodegib plasma concentration. (DOCX 24 kb)

Supplementary material & methods.

Regulation of megakaryocytic factors by CALR mutants in murine and human cell lines. a Detection of Fli-1 and Ets-1 or b Gata1 mRNA expression by RT-qPCR in the indicated 32D and HL60e cells. The experiments were performed in triplicates. Mean and SD are indicated. ***P

Expression of the C-terminally YFP-tagged CALR del52 mutant leads to STAT5 phosphorylation and cytokine-independent growth of 32D MPL cells. a 32D cells stably transduced with WT CALR-flag-YFP, CALR del52-flag-YFP, or empty vector (EV) −/+ MPL were WEHI-starved for 16 h and protein lysates were prepared. SDS-PAGE and Western blotting was performed and the indicated antibodies were used for immunodetection. YFP was detected using a GFP-specific antibody, and GAPDH served as loading control. b The indicated 32D MPL cells were cultured in WEHI-free medium (2 × 105 cells/ml). The cells were counted every 24 h for 3 days. The cell counts are mean values of triplicates. (PDF 53 kb)

Mutant CALR grants factor-independence, protects from apoptosis, and activates downstream signaling in an MPL-dependent manner. a A proliferation assay was performed with the indicated cell lines (2 × 105 cells/ml). 32D MPL cells were counted every 24 h for 4 days. The cell counts are mean values of triplicates. b Empty vector, WT CALR, CALR del52, and ins5 expressing 32D cells were seeded in a density of 5 × 105 cells/ml and grown for 48 h in WEHI-free medium. Apoptosis was analyzed by flow cytometry after staining with Annexin V-APC and 7-AAD. Mean and SD are indicated. *P

Missing CALR-mutant detection in Western blotting is not due to loss of the antibody epitope or an SDS-PAGE artifact. a HEK293T cells were transiently transfected with expression vectors for flag-tagged WT CALR, CALR del52, a C-terminal truncation mutant (ΔC; aa R366) and empty vector. Twenty-four hours after, transfection lysates were generated and SDS-PAGE followed by Western blotting were performed. The indicated antibodies were used for immunostaining. The used CALR antibody bound to the CALR ΔC mutant. b Native page followed by Western blotting was performed with non-denatured protein lysates of 32D CALR cells. The PVDF membrane was stained with CALR antibody, and the membrane was stripped and stained with a flag-specific antibody. (PDF 67 kb)

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

Summary Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.