Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival

<p>Abstract</p> <p>Background</p> <p>The JAK2^V617Fmutation plays a major role in the pathogenesis of myeloproliferative neoplasms and is found in the vast majority of patients suffering from polycythemia vera and in roughly every second patient suffering from essential thrombocythemia or from primary myelofibrosis. The V617F mutation is thought to provide hematopoietic stem cells and myeloid progenitors with a survival and proliferation advantage. It has previously been shown that activated JAK2 promotes cell survival by upregulating the anti-apoptotic STAT5 target gene Bcl-xL. In this study, we have investigated the role of additional apoptotic players, the pro-apoptotic protein Bim as well as the anti-apoptotic protein Mcl-1.</p> <p>Methods</p> <p>Pharmacological inhibition of JAK2/STAT5 signaling in JAK2^V617Fmutant SET-2 and MB-02 cells was used to study effects on signaling, cell proliferation and apoptosis by Western blot analysis, WST-1 proliferation assays and flow cytometry. Cells were transfected with siRNA oligos to deplete candidate pro- and anti-apoptotic proteins. Co-immunoprecipitation assays were performed to assess the impact of JAK2 inhibition on complexes of pro- and anti-apoptotic proteins.</p> <p>Results</p> <p>Treatment of JAK2^V617Fmutant cell lines with a JAK2 inhibitor was found to trigger Bim activation. Furthermore, Bim depletion by RNAi suppressed JAK2 inhibitor-induced cell death. Bim activation following JAK2 inhibition led to enhanced sequestration of Mcl-1, besides Bcl-xL. Importantly, Mcl-1 depletion by RNAi was sufficient to compromise JAK2^V617Fmutant cell viability and sensitized the cells to JAK2 inhibition.</p> <p>Conclusions</p> <p>We conclude that Bim and Mcl-1 have key opposing roles in regulating JAK2^V617Fcell survival and propose that inactivation of aberrant JAK2 signaling leads to changes in Bim complexes that trigger cell death. Thus, further preclinical evaluation of combinations of JAK2 inhibitors with Bcl-2 family antagonists that also tackle Mcl-1, besides Bcl-xL, is warranted to assess the therapeutic potential for the treatment of chronic myeloproliferative neoplasms.</p

Identification and characterization of preclinical models of spliceosome mutant myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are the most common myeloid malignancies of the elderly, characterized by dysplasia and ineffective haematopoiesis. Approximately 25.000 new cases occurring in the EU annually and 30-40% of MDS patients eventually progress acute myeloid leukaemia (AML). The disease (MDS) is still considered to be understudied. However, recent studies using whole exome sequencing (WES) analysis have led to the discovery of recurrent somatic mutations in genes, such as SF3B1 and SRSF2 functioning in the subunit of the spliceosomal machinery in MDS patients. The evidence to date points to heterozygous mutations in the spliceosome machinery components and the mutations are often clustered in particular functional domains, indicating an altered gain-of-function, and underscoring patho-biological relevance. Spliceosomal mutations may lead to miss-splicing and ultimately decreased expression of intact tumor suppressor genes. We hypothesize, that SRSF2 and SF3B1 skew hematopoietic cell differentiation and promote transformation due to miss-splicing of transcripts. To address this, I stably transfected cytokine dependent TF-1 AML M6 cells with a cDNA of SRSF2 encoding either the wt or mutant (SRSF2P95L) gene. Accordingly, I selected clones of SRSF2P95L, growing in a cytokine independent manner, for further characterization to test the impact of the SRSF2P95L mutation on the oncogenic phenotype. As a complementary approach, I aimed to study the impact of the allele- specific inactivation of endogenous SF3B1K666N by using the CRISPR/Cas9 degron knock-in approach.Identification and characterization of preclinical models of spliceosome mutant myelodysplastic syndrome

JAK2/STAT5 Inhibition Circumvents Resistance to PI3K/mTOR Blockade: A Rationale for Cotargeting These Pathways in Metastatic Breast Cancer

SummaryHyperactive PI3K/mTOR signaling is prevalent in human malignancies and its inhibition has potent antitumor consequences. Unfortunately, single-agent targeted cancer therapy is usually short-lived. We have discovered a JAK2/STAT5-evoked positive feedback loop that dampens the efficacy of PI3K/mTOR inhibition. Mechanistically, PI3K/mTOR inhibition increased IRS1-dependent activation of JAK2/STAT5 and secretion of IL-8 in several cell lines and primary breast tumors. Genetic or pharmacological inhibition of JAK2 abrogated this feedback loop and combined PI3K/mTOR and JAK2 inhibition synergistically reduced cancer cell number and tumor growth, decreased tumor seeding and metastasis, and also increased overall survival of the animals. Our results provide a rationale for combined targeting of the PI3K/mTOR and JAK2/STAT5 pathways in triple-negative breast cancer, a particularly aggressive and currently incurable disease

Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model

To establish a pre-clinical animal model for testing drugs with potential effects on MPN, we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2 knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelets and granulocyte levels. In a transplantation assay, JAK2-V617F positive bone marrow cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea. Hydroxyurea led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized red cell and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced phospho-Stat5 levels, whereas hydroxyurea strongly increased phospho-Stat5, most likely due to the elevated erythropoietin levels in response to the hydroxyurea induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of hydroxyurea and represents an adverse effect that should be avoide

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease”

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

JAK1/2 and pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease

JAK inhibitors have demonstrated rapid and durable reductions in splenomegaly, as well as improvement in symptoms and quality of life in patients with myelofibrosis. However, the impact on the mutant allele burden and bone marrow fibrosis has been modest, indicating that combinations with other agents may further improve outcomes. Histone deacetylase inhibition has emerged as a promising combination modality based on in vitro studies using JAK2V617F mutant models that suggested a synergistic effect upon combination with a JAK2 inhibitor, and encouraging single-agent activity of the pan-deacetylase inhibitor panobinostat in phase I/II myelofibrosis trials. Here, we investigated the combination of the JAK1/2 inhibitor ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. The combination was found to have a more profound effect on efficacy readouts as compared to either agent alone, and the analysis of pharmacodynamic readouts demonstrated that ruxolitinib and panobinostat have non-overlapping and complementary effects on biological pathways

Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings

Abstract to be written, for journal. Please find the synopsis for OAK reviewers only below: In this publication we disclose the discovery of DCAF1 E3 ligase binding matter and the functionalization to PROTACs for TPD of BRD9, Tyrosine kinases (Dasatinib) and BTK. A lot of data has been initiated as part of the work by the ONC9LIG team (I was an integral part of). Project work on DCAF1 has been discontinued roughly 6-7 years ago (focus on BTK bifunctional degraders as ONC9BTK team). All compounds (IP inventory) have been shared with GDC in a previous email and the proposal to publish DCAF1 binders as well as the DCAF1-degraders was formally approved in August 2021 by the TPD steering committee including Jay and Karin. We disclose 5 key messages: • Discovery of DCAF1 as an essential ligase (DepMAP) and hypothesis to overcome CBRN mediated resistance. Leads into non-covalent DCAF1 binder discovery and characterisation, which can be functionalized for TPD for PROTACs (Figure 1) • With a first degrader prototype we do show that endogenous mainly nuclear ligase DCAF1 can degrade BRD9 (BRD9-PROTAC, another nuclear protein. Key message is around in-depth chemical and genetic validation (Figure 2) • We expand the degradation space with a second more promiscuous degrader prototype against tyrosine kinases (Dasatinib-DCAF1 PROTAC) and show that DCAF1 can even tackle cytoplasmic as well as membrane bound tyrosine kinases (Figure 3) – we touch here also on the tox observation • We expand this further to an ONC target BTK with more specific PROTACs (linear and more complex linkers) that we characterize in-depth (Figure 4), here we describe a very systematic in vitro approach and show that unfortunately there is not a simple correlation between in vitro findings (complex, ubi etc.) and cellular degradation potency (as seen by many but not really worked up so systematically). Here we build up the path towards discovery of our most potent DCAF1-BTK degrader (JRF) • JRF449 characterization and ultimate rescue experiment in CBRN-BTK PROTAC resistant cells where we do see BTK degradation and effect in proliferation with a DCAF1-BTK degrader even in CRBN resistant settings (in finalization) (draft Figure 5) Key internal flags towards DCAF1 as an alternative ligase that led to termination were and still are: • Discovery of only 1 chemical series (to be published in accompanying manuscript OAK 49705) • This chemical series shows pan toxicity at >5uM (CLiP studies >300 cell lines tested revealed this) o Rescue experiments to understand if this is on-target toxicity remained unsuccessful (Focused mutagenesis around donut-hole binding site, whole gene Variomics, endogenous gene editing around donut-hole locus) o Until now tough we do not have a clear answer if this is off or on-target tox (DCAF1 is an essential ligase) • We comment on these observations of tox briefly in the manuscript • In addition, even tough we had extensive efforts on DCAF1-BTK degraders (50+ molecules) we never reached a potency range compared to CRBN-BTK (in-house) or (VHL, external). Our best degrader JRF449 (in publication has a DC50 of about 100nM) o Literature also suggests that DCAF1 is somehow in a tetrameric self-inhibitory mode We have had discussions with GDC colleagues concerning the chemical matter to be published in this manuscript and got verbal consent that the compounds can be disclosed. GDC decided not to patent the DCAF1 binders on their own and all the other elements of the PROTACs in this manuscript, thelinkers and war-heads are covered by own patents (BTK-binders and linkers) or in the public domain (BRD9 binder and Dasatinib)