ALBERT

Description: Introduction: PcG proteins form two main multiprotein complexes, Polycomb repressive complex 1 (PRC1) and PRC2. They repress the transcription of target genes. Polycomb group ring finger protein1 (PCGF1) is a component of PRC1.1, a non-canonical PRC1.1 that monoubiquitylates H2A at lysine 119 in a manner independent of H3K27me3. Several groups including ours showed that the loss of Ezh2, a component of PRC2, promotes the development of JAK2 V617F-induced Myelofibrosis (MF) in mice. However, the role of PRC1.1 in hematologic malignancies is still not fully understood. We found that the deletion of PCGF1 in mice promotes myeloid commitment of hematopoietic stem and progenitor cells (HSPCs), and eventually induces a lethal myeloproliferative neoplasm (MPN)-like disease in mice (Nakajima-Takagi Y, unpublished data). Based on these findings, we investigated the role of PCGF1 in a mouse model of JAK2V617F-induced myelofibrosis. Methods: We transplanted BM cells from Cre-ERT2, PCGF1flox/flox;Cre-ERT2, JAK2V617F;Cre-ERT2, and JAK2V617F;PCGF1flox/flox;Cre-ERT2 mice into lethally irradiated recipient mice. We deleted PCGF1 by tamoxifen administration 4 weeks after transplantation. Results: JAK2/PCGF1 KO mice developed lethal MF significantly earlier than the other genotypes (p

Description: Background:Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine synthesis, the conversion of DHO to orotate. DHODH inhibition has been described recently as a new approach for treating acute myeloid leukemia (AML) by inducing differentiation of diverse AML subclasses. PTC299 represents a novel potent DHODH inhibitor and recently clinical development of PTC299 as a potential treatment option for acute leukemia was initiated. Here, we explored the efficacy of DHODH inhibitor PTC299 for MDS. Methods:Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. PTC299 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometric analysis and RNA-sequencing (RNA-seq). Gene expression levels were analyzed by quantitative PCR (qPCR). Results:PTC299 inhibited proliferation of AML cell lines and induced their differentiation. As previously reported in other DHODH inhibitors, upregulation of CD11b was observed after PTC299 treatment in both HL-60 and THP-1 cells. In addition, PTC299 inhibited the proliferation of MDS cell lines, MDSL and SKM-1 cells, with EC50s of 12.6 nM in MDSL cells and 19.7 nM in SKM-1 cells. The inhibitory effect was reversed by the exogenous addition of 100 µM uridine, which bypasses the requirement for de novo pyrimidine synthesis by feeding into the salvage pathway, thereby negating the need for DHODH. Because the basal expression levels of CD11b are high in MDS cells, we examined the expression levels of CD38. Both cell lines showed dose-dependent upregulation of CD38 after PTC299 treatment. To investigate the possible synergism between PTC299 and decitabine, we treated MDSL and SKM-1 cells with increasing concentrations of PTC299 and decitabine as single agents or in combination. After 3 days of culture, cells were analyzed by MTS assays. PTC299 and decitabine exerted a enhanced cytotoxic effect on MDSL and SKM-1 cells. Similar results were obtained with primary MDS samples. In Annexin/PI assays, the percentage of apoptotic cells was increased by combination of PTC299 with decitabine in both cell lines. Mechanistically, treatment with PTC299 induced an intra-S-phase arrestfollowed by entry intoapoptotic cell death. It has also been reported that the expression of p53 is increased in response to the intra-S-phase arrest. To understand the genome-wide effects and target genes of PTC299 and the combination with decitabine, we performed RNA-seq of MDSL and SKM-1 cells treated with PTC299, decitabine, or the combination of both agents versus DMSO-treated cells. Gene set enrichment analysis (GSEA) using our RNA-seq data confirmed that MYC target gene sets were negatively enriched in both PTC299-, decitabine- and combination- treated cells. KEGG pathway enrichment analysis revealed activation of genes associated with apoptosis in both cell lines. To better elucidate a synergistic effect of PTC299 and decitabine, we performed qPCR of CDKN1A, which is a major target of p53 activity. The mRNA expression levels of CDKN1A were upregulated after treatment with PTC299, which was further enhanced by the combination with decitabine. Conclusions:Our result indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells in vitro and acts in at least an additive and possibly synergistic manner with decitabine in this process. This combination therapy could be a new therapeutic option for the treatment of MDS. Disclosures Lennox: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Sheedy:PTC Therapeutics: Employment.

Description: BCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (BcorΔE4/y) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (BcorΔE9-10/y), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. BcorΔE9-10/y mice developed lethal T-ALL in a similar manner to BcorΔE4/y mice, whereas BcorΔE9-10/y hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2. Tet2Δ/ΔBcorΔE9-10/y mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2Δ/ΔBcorΔE9-10/y MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in BcorΔE9-10/y progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

Description: Background: Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Tubulin belongs to protein superfamily of globular proteins. Monomeric tubulin can polymerize into microtubules, which play an important role in the attachment and segregation of chromosomes in various phases of cell division. Therefore, the targeting of microtubules represents a therapeutic strategy against both solid and hematological cancers. The first approved tubulin binding agent by the FDA was vincristine, which has been clinically used to treat multiple types of cancers, particularly hematological malignancies. Over the past few decades, additional tubulin binding agents have been developed and received FDA approval, mostly for applications to cancer therapy. These agents have been classified by their binding sites on tubulin, which influences their roles in the inhibition or stabilization of polymerized microtubules. We herein investigated the efficacy of PTC-028, a novel microtubule polymerization inhibitor for MDS. Method: Anti-MDS efficacy of PTC-028 was studied using human MDS cell lines and primary MDS cells in vitro. The efficacy of PTC-028 was also assessed in a xenograft mouse model of MDS using an MDS cell lines. PTC-028 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometry and RNA sequencing. Results: A previous study reported that PTC596 suppressed cell proliferation and induced apoptosis in AML cell lines. Since MDS is regarded as a pre-leukemic stage, we examined the effects of PTC-028, another novel microtubule polymerization inhibitor, on MDS cells. PTC-028 induced a dose-dependent inhibition of cell proliferation on MDS cell lines, such as MDS-L and SKM-1 cells. Caspase 3/7 activity was also significantly induced in MDS-L and SKM-1 cells in the presence of PTC-028, suggesting the induction of apoptotic cell death by PTC-028. We then isolated CD34+ cells from primary MDS BM samples and investigated the efficacy of PTC-028 on primary MDS cells. The efficacy of PTC-028 in CD34+ MDS cells was also confirmed by cell proliferation assays. To enhance the therapeutic benefit of PTC-028 on MDS cells, we investigated synergism between PTC-028 and DNA hypomethylating agents. We treated MDS-L and SKM-1 cells with PTC-028 in combination with DNA hypomethylating agents. After 3 days of culture, cell growth was analyzed by MTS and Annexin V assays. PTC-028 synergized with hypomethylating agents, such as decitabine and azacitidine, to inhibit the growth and induce apoptosis of MDS cells. We assessed the efficacy of PTC-028 in a xenograft mouse model of MDS using an MDS cell line. Recipient mice were treated with PTC-028 for 7 weeks. PTC-028 significantly inhibited the growth of MDS-L cells and prolonged the overall survival of recipient mice. Furthermore, a significant synergistic effect was observed between PTC-028 and decitabine. Mice that received combination therapy showed moderate weight loss 11 days after the initiation of the treatment, but subsequently recovered. Mechanistically, a treatment with PTC-028 induced G2/M arrest followed by apoptotic cell death. We then investigated the effect of PTC-028 on the levels of soluble (unpolymerized) versus polymerized tubulin in MDS-L cells. Cells were treated with PTC-028 and paclitaxel for 4 hours, and cell lysates were then separated into soluble and polymerized fractions by centrifugation. The visualization of tubulin fractions by Western blotting demonstrated that the PTC-028 treatment for 4 hours resulted in the near-complete loss of polymerized microtubules. In contrast, polymerized microtubules increased in cells treated with paclitaxel, which stabilizes microtubules against depolymerization. These result indicates that PTC-028 also acts as a microtubule polymerization inhibitor. Conclusion: Our data reveal a possible chemotherapeutic strategy for PTC-028 and PTC-596 in MDS by disruption of microtubule dynamics as a single agent and in combination with hypomethylating agents. The present study provides a preclinical framework for the clinical evaluation of this promising therapeutic approach to improve outcomes in MDS patients. Disclosures Lennox: PTC therapeutics: Current Employment. Sheedy:PTC therapeutics: Current Employment. Weetall:PTC Therapeutic: Current Employment.