Cohesin-dependent regulation of gene expression during differentiation is lost in cohesin-mutated myeloid malignancies.

Cohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies

Development of resistance to type II JAK2 inhibitors in MPN depends on AXL kinase and is targetable.

PURPOSE Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Since clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. METHODS MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC-/RNA-sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NSG mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Since inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL-MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL-MAPK. CONCLUSIONS We report on a novel mechanism of AXL-MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN

Genetics of Myeloproliferative Neoplasms.

Myeloproliferative neoplasms are hematopoietic stem cell disorders based on somatic mutations in JAK2, calreticulin, or MPL activating JAK-STAT signaling. Modern sequencing efforts have revealed the genomic landscape of myeloproliferative neoplasms with additional genetic alterations mainly in epigenetic modifiers and splicing factors. High molecular risk mutations with adverse outcomes have been identified and clonal evolution may promote progression to fibrosis and acute myeloid leukemia. JAK2V617F is recurrently detected in clonal hematopoiesis of indeterminate potential with increased risk for vascular events. Insights into the genetics of myeloproliferative neoplasms has facilitated diagnosis and prognostication and poses novel candidates for targeted therapeutic intervention

Role of unphosphorylated STAT5 in maintenance of Acute Myeloid Leukemia cells

Background In its phosphorylated state, the Signal Transducer and Activator of Transcription 5 (STAT5) A and B form homo- or heterodimers, which bind to chromatin and activate expression of target genes. In leukemia models harboring FLT3-ITD, BCR-ABL or JAK2 mutations, constitutive phosphorylation of STAT5 activates key proliferation and survival transcriptional programs. Recent studies in mouse hematopoietic progenitor cells suggest a distinct function of unphosphorylated STAT5 (uSTAT5): via restricting the access of ERG to target genes uSTAT5 acts as a repressor of megakaryocytic transcriptional programs [1]. The goal of this study is to examine the biological role of uSTAT5A and B in acute myeloid leukemia (AML) and to explore their role as potential therapeutic targets. Results We initially screened a panel of human AML cell lines and patient samples for STAT5A and STAT5B expression and phosphorylation at defined tyrosine residues. Most of the samples displayed strong expression of both STAT5A and STAT5B. Phosphorylation of STAT5 proteins at tyrosine 694 (pSTAT5A) and 699 (pSTAT5B) residues was strongly dependent on the presence of FLT3-ITD mutations. To explore the role of uSTAT5A/B, we performed doxycycline-inducible, short-hairpin RNA (shRNA) mediated knock-down of STAT5A and STAT5B. Targeting STAT5A or STAT5B severely suppressed cell proliferation across the entire tested panel; nevertheless, differentiation assays revealed that only the suppression of uSTAT5B induced cellular differentiation. In line, gene expression profiling by high-throughput sequencing (RNA-seq), demonstrated enrichment of monocytic differentiation programs in the THP-1 cell line upon loss of uSTAT5B. To further assess the distinct effects of STAT5A and STAT5B, we performed SILAC-based mass spectrometry and identified several STAT5 interacting partners in AML cell lines. While uSTAT5A primarily was found to be associated with proteins involved in RNA processing and translation initiation pathways, uSTAT5B coprecipitated chromatin- and histone-binding proteins, such as the transcription factor ETV6 or the histone H3K4 demethylase KDM5C. Finally, to elucidate the role of STAT5 on leukemic cell function in a mouse model, Stat5fl/fl or Stat5fl/fl_Mx1-Cre bone marrow cells were transformed with a retroviral construct of Mll/Af9 and transplanted into lethally irradiated mice. In the second round of transplantations, we performed a pIpC induction of Mx-1-Cre recombinase to explore, whether complete excision of Stat5 can prevent leukemia development. Surprisingly, animals transplanted with Stat5-depleted MA9 cells died briefly after excision with median survival of 15 days, while median survival of the Stat5fl/fl control was 26 days. Bone marrow cells from both cohorts were investigated for the expression of CD11b and Gr-1, both markers of differentiated hematopoietic cells. Interestingly, the Stat5fl/fl_Mx1-Cre-MA9-group, depleted of Stat5, showed a strong increase in the double-positive cells compared to control group suggesting a more mature phenotype. Summary In summary, our data indicate that uSTAT5B is involved in the regulation of differentiation through modulation of the epigenetic landscape and transcriptional programs of leukemic cells. Targeting of uSTAT5B or its downstream pathways might represent an interesting novel strategy in AML treatment.HINTERGRUND Signal Transducer and Activator of Transcription (STAT) Proteine sind essentielle Mediatoren von externen Signalen in den Zellkern. Insgesamt können 7 Mitglieder unterschieden werden (STAT1-6), wobei für STAT5 die Isoformen STAT5A und -B existieren. Nach Aktivierung von Rezeptoren der Zelloberfläche kommt es zur Phosphorylierung von STAT5A und -B, zur Bildung von Homo- oder Heterodimeren und einer Translokation in den Nukleus, wo dann ein transkriptionelles Programm initiiert wird. Kürzlich konnte in murinen hämatopoetischen Vorläuferzellen gezeigt werden, dass nicht-phosphoryliertes STAT5B (uSTAT5B) ein ERG-induziertes Programm im Rahmen der megakaryozytären Differenzierung unterdrücken kann, also als transkriptioneller Repressor fungiert. Das Ziel dieser Arbeit war die Rolle von uSTAT5 im Rahmen der Erkrankung akute myeloische Leukämie (AML) sowie als therapeutische Zielstruktur zu untersuchen. ERGEBNISSE Wir konnten zeigen, dass STAT5A und -B in AML-Zellen sowohl in phosphorylierter als auch nicht-phosphorylierter Form vorliegt, und der Status der Phosphorylierung im Wesentlichen von der Co-Expression der mutierten Tyrosinkinase FLT3-ITD abhängt. Um die Bedeutung von uSTAT5 näher zu untersuchen, wurde die Expression von STAT5A oder -B mittels einer Doxyzyklin-induzierbaren shRNA supprimiert. Nach Knockdown von STAT5A und -B zeigte sich eine signifikante Reduktion der Proliferationsrate und insbesondere nach STAT5B-Knockdown die Aktivierung eines transkriptionellen Differenzierungsprogramms (Nachweis mittels RNA-Sequenzierung), gefolgt von der Expression von CD11b und morphologischen Veränderungen. Um den Mechanismus dieser Veränderungen zu untersuchen, wurden THP1, SKM1 und MV4-11 AML-Zellen in SILAC-Medium kultiviert und das Interaktom von STAT5A und -B mittels pull-down-Experimenten und nachfolgender Massenspektrometrie untersucht. Interessanterweise zeigten sich für uSTAT5A und uSTAT5B unterschiedliche Interaktionsprofile. Während uSTAT5A vor allem mit Proteinen der RNA-Prozessierung und Translation-Initiierung interagierte, war uSTAT5B überwiegend an Chromatin- und Histon-bindende Proteine, wie z.B. ETV6 oder KDM5C, gebunden. Um die Rolle von STAT5 in einem Leukämiemausmodell zu untersuchen, wurden Knochenmarkzellen einer Stat5fl/fl oder einer Stat5fl/fl_Mx1-Cre Maus mit dem Konstrukt MSCV_Mll/Af9_IRES-GFP infiziert und in lethal-bestrahlte Empfängertiere transplantiert. Nach Depletion von Stat5 ergab sich in sekundär transplantierten Mäusen eine signifikante verminderte Überlebenszeit im Vergleich zur Kontrollmäusen. Die genauere Untersuchung der Leukämiezellen zeigte eine zunehmende Ausreifung der Stat5-depletierten Tiere. Dieses Ergebnis weist auf ein Differenzierungssyndrom hin, wie es auch bei akuter Promyelozyten-Leukämie nach ATRA-Behandlung beobachtet wird und ist in Einklang mit der in Zelllinien beobachteter Differenzierung nach STAT5B-Knockdown. ZUSAMMENFASSUNG In dieser Arbeit konnten wir zeigen, dass neben phosphoryliertem STAT5 auch uSTAT5A und- B definierte biologische Funktionen regulieren. Insbesondere uSTAT5B scheint durch Modulation von epigenetischen Regulatoren die Differenzierung von AML-Zellen zu unterdrücken und repräsentiert einen neuen therapeutischen Ansatz in der Behandlung von AML-Patienten

Dual targeting of JAK2 and ERK interferes with the myeloproliferative neoplasm clone and enhances therapeutic efficacy.

Myeloproliferative neoplasms (MPN) show dysregulated JAK2 signaling. JAK2 inhibitors provide clinical benefits, but compensatory activation of MAPK pathway signaling impedes efficacy. We hypothesized that dual targeting of JAK2 and ERK1/2 could enhance clone control and therapeutic efficacy. We employed genetic and pharmacologic targeting of ERK1/2 in Jak2V617F MPN mice, cells and patient clinical isolates. Competitive transplantations of Jak2V617F vs. wild-type bone marrow (BM) showed that ERK1/2 deficiency in hematopoiesis mitigated MPN features and reduced the Jak2V617F clone in blood and hematopoietic progenitor compartments. ERK1/2 ablation combined with JAK2 inhibition suppressed MAPK transcriptional programs, normalized cytoses and promoted clone control suggesting dual JAK2/ERK1/2 targeting as enhanced corrective approach. Combined pharmacologic JAK2/ERK1/2 inhibition with ruxolitinib and ERK inhibitors reduced proliferation of Jak2V617F cells and corrected erythrocytosis and splenomegaly of Jak2V617F MPN mice. Longer-term treatment was able to induce clone reductions. BM fibrosis was significantly decreased in MPLW515L-driven MPN to an extent not seen with JAK2 inhibitor monotherapy. Colony formation from JAK2V617F patients' CD34+ blood and BM was dose-dependently inhibited by combined JAK2/ERK1/2 inhibition in PV, ET, and MF subsets. Overall, we observed that dual targeting of JAK2 and ERK1/2 was able to enhance therapeutic efficacy suggesting a novel treatment approach for MPN

Co-Occurring CSF3R W791* Germline and Somatic T618I Driver Mutations Induce Early CNL and Clonal Progression to Mixed Phenotype Acute Leukemia

Chronic neutrophilic leukemia (CNL) relates to mutational CSF3R activation with mem-brane proximal CSF3R mutations such as T618I as driver mutations, but the significance of truncat-ing mutations is not clarified. In CNL, concomitant mutations promote disease progression, but insight into longitudinal acquisition is incomplete. In this study, we investigated the role of co-oc-curring germline and somatic CSF3R mutations in CNL, and assessed the impact of clonal evolution on transformation to acute leukemia. We employed sequential next generation sequencing and SNP array karyotyping to assess clonal evolution in CNL of early manifestation age based on a 33-year-old patient. Germline vs. somatic mutations were differentiated using a sample from the hair folli-cle. To investigate a potential predisposition for CNL development and progression by germline CSF3R-W791*, allelic localizations were evaluated. We detected a somatic CSF3R-T618I mutation at 46% variant allele frequency (VAF) at the time of CNL diagnosis, which co-occurred with a CSF3R-W791* truncation at 50% VAF in the germline. Evaluation of allelic localization revealed CSF3R-T618I and W791* on the same allele. A concomitant ASXL1 mutation at 39% VAF increased to 48% VAF upon transformation to mixed phenotype acute leukemia (MPAL), which has both myeloid and lymphoid features. Clonal evolution further involved expansion of the CSF3R double-mutant clone to 90% VAF via copy neutral loss of heterozygosity on chromosome 1p and the emergence of a RUNX1 mutant subclone. Allogeneic transplantation induced complete remission. This study highlights that CNL not only transforms to AML but also to MPAL. The molecular evolution is especially interesting with a CSF3R-W791* mutation in the germline and acquisition of CSF3R-T618I on the same allele compatible with increased susceptibility for mutation acquisition facilitating RUNX1-related clonal transformation