Apoptosis Of Hematopoietic Cells Research Articles

A Phase 1 Study of TP-3654, an Orally-Delivered PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis

Background: Myelofibrosis (MF) is characterized by bone marrow fibrosis (BMF) and ineffective extramedullary hematopoiesis resulting in splenomegaly and debilitating symptoms. An activating Janus kinase 2 (JAK2) mutation (V617F) has been frequently observed in MF. Ruxolitinib (a JAK1/JAK2 inhibitor) reduces splenomegaly and improves constitutional symptoms, but appears to offer a modest reduction of BMF. Patients with MF who are intolerant to JAK inhibitors or their components, or for whom JAK inhibitors have failed, have limited treatment options. The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine kinases are overexpressed in hematological malignancies. Because PIM kinase expression is regulated by JAK2 signaling, PIM kinase inhibition is a potential therapeutic target for JAK2 mutant-driven malignancies, such as MF. TP-3654, an investigational agent, has been shown to reduce proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone reduced leukocytosis and spleen size in an in vivo murine model of JAK2V617F-induced MF, as well as an apparent reduction of BMF. A phase 1 study is being conducted to evaluate TP-3654 monotherapy in patients with MF. Study Design and Methods: This phase 1, multicenter, dose-escalation, open-label study is evaluating TP-3654 monotherapy in patients with MF who previously failed a JAK inhibitor or who are ineligible to receive ruxolitinib or fedratinib (NCT04176198). Primary objectives are to determine the incidence of dose-limiting toxicities (DLT) at escalated doses of TP-3654 and treatment emergent adverse events. Secondary objectives are to determine QT interval changes, establish the pharmacokinetic profile, and assess preliminary disease activity. Exploratory objectives are pharmacodynamic markers in peripheral blood and bone marrow biopsy samples. Eligible patients have a primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF) based on the World Health Organization diagnostic criteria and intermediate-2 or high-risk primary or secondary MF based on the Dynamic International Prognostic Scoring System; previously failed, or are ineligible to receive, treatment with a JAK inhibitor; grade ≥2 MF, as confirmed by BM biopsy ≤12 weeks prior to screening; have a platelet count >50x109/L, absolute neutrophil count ≥1x109/L, hemoglobin level ≥8 g/L, peripheral blood blast counts <10%, Eastern Cooperative Oncology Group performance status ≤2, life expectancy ≥3 months, and adequate renal and hepatic function; have spleen length ≥5 cm by palpation or spleen volume ≥450 cm3 by computerized tomography/magnetic resonance imaging, and show ≥2 measurable symptoms per the MF Symptom Assessment Form, version 4.0. Patients must not have received prior systemic antineoplastic therapy or any experimental therapy within 14 days or 5 half-lives before TP-3654; had major surgery ≤2 weeks before first study dose; have had splenic irradiation ≤6 months prior to screening; have acute myeloid leukemia or myelodysplastic syndrome; or had prior stem cell transplantation (SCT) or be eligible for allogeneic bone marrow or SCT. Enrollment of approximately 50 patients is planned. Patients will receive oral TP-3654. Dose-escalation will be performed using a Bayesian logistic regression model with escalation with overdose control. Adverse events occurring during the first cycle will be considered in the determination of the maximum tolerated dose (MTD). Dose escalation will continue until identification of the MTD or a suitable recommended phase 2 dose. This study is currently recruiting patients. Disclosures Lebedinsky: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Anthony:Exact Sciences: Consultancy; Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Mohi:Tolero Pharmaceuticals Inc.: Research Funding. Yang:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Mei:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Braendle:Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment.

Effects of Genetic Variation on Urinary Small Molecule Signatures of Mice after Exposure to Ionizing Radiation: A Study of p53 Deficiency.

Due to risks from potential exposures to ionizing radiation (IR), improved radiological countermeasures are required, as well as rapid high-throughput biodosimetry. Genotypic variation in the general population contributes to differences in radiosensitivity that may affect biodosimetry accuracy. Previous studies utilized radiosensitive mutant mouse models (Parp1−/− and Atm−/−) to determine the effects of genotypic deficiency on radiation signatures. Here, we extend this approach by examining changes in the urinary metabolome in a hematopoietic (HP) resistant mouse model (p53−/−) after IR exposure. As p53 is a primary regulator in radiation response and apoptosis, limited hematopoietic stem cell apoptosis leads to reduced mortality at doses of ~8–10 Gy but increased mortality at higher doses (>15 Gy) due to mitotic catastrophe in gastrointestinal (GI) crypt cells. Urine was collected from mice (wild-type (WT), p53+/−, and p53−/−) pre-irradiation and at 4 and 24 h after total body irradiation (TBI) (WT: 8 and 10 Gy; p53−/−: 10 Gy) for metabolic phenotyping using an ultra-performance liquid chromatography mass spectrometry (UPLC-MS) platform. Minimal differences were detected between unirradiated WT, p53+/−, and p53−/− mice. While similar perturbations were observed for metabolites involved in tryptophan, vitamin B6, and histamine pathways, glycine conjugation, and redox metabolism for WT and p53−/− mice after TBI, an overall dampened response was observed in p53-deficient mice. Despite comparable metabolite patterns between genotypes, differentiation was achieved through receiver operating characteristic curve analysis with high specificity and sensitivity for carnitine, N1-acetylspermidine, and creatine. These studies highlight that both attenuated and dampened metabolic responses due to genetic variability in the general population need to be addressed in biodosimetry frameworks.

Short-term culture under pressurized and hypoxic conditions reduces hematopoietic stem cell apoptosis and improves cellular function of transplant products

Background & Aim Hematopoietic stem cell transplantation (HSCT) is a potential curative therapy for numerous diseases. The success of HSCT is dependent upon the viability, homing, and engraftment capabilities of the hematopoietic stem cells (HSCs). Current clinical approaches allow for storage times up to 48 hours at either room temperature (20-24°C) or refrigerated conditions (2-8°C) prior to transplantation, resulting in high levels of cellular apoptosis and reduced functionality, thus compromising the effectiveness of the transplanted cells. To address this, we substituted the current conditions with short-term culturing methods ( Methods, Results & Conclusion Environmental optimization can selectively enhance the fitness and functionality of specific cell types and increase the stemness or differentiation potential of cells in culture. By varying the temperature (4°C, room temperature, 37°C, 43°C), oxygen (20%, 5%, and 1%), and pressure levels (1.00 to 1.35 atm), we optimized the culture conditions to improve the functionality of HSCs. Cell health was measured using assays measuring apoptosis and metabolic function. Significant decreases in cell populations positive for apoptosis markers, annexin V and DilC1(5), were observed after 24 hours of incubation in optimized conditions compared to current clinical approaches. DUSP4, a stress-related gene that prevents apoptosis, was upregulated under optimized environmental conditions; this, along with higher ATP levels, is indicative of functional and healthy cells. Preconditioned cells exhibit characteristics of a product with improved rates of bone marrow homing and immune reconstitution. They show increased surface expression of the receptor for stromal cell-derived factor (CXCR4), the sialomucin receptor for P-selectin glycoprotein ligand-1 (PSGL-1), the β1 integrin very-late antigen (VLA-4), and the tetraspanin CD82, which are all implicated in the homing capacity of HSCs to the bone marrow. Immune reconstitution, as assessed by measuring proliferation and differentiation via a colony forming units in vitro, predicts robust reconstitution capability of HSCs cultured under optimized conditions. As shown, cells for HSCT maintained under optimized temperature, oxygen, and pressure levels result in improved cell viability and function over conventional storage conditions, suggesting a potential clinical benefit in the transplantation setting.

Protective effect of bleomycin on 5-azacitidine induced cytotoxicity and apoptosis in mice hematopoietic stem cells via Bcl-2/Bax and HMGB1 signaling pathway

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC50 value of 16 μM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC50 of 64 μM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy.

P53 Mediated Bone Marrow Mesenchymal Stem Cell Expansion Supports Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p<0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

Astragalus Polysaccharide Promotes Hematopoiesis in an Irradiated Mouse Model and Reduces Apoptosis of Hematopoietic Cells

Background: Huangqi (the dried root of Radix Astragali) is a notable Chinese herb that has been used to promote hematopoiesis for centuries. Astragalus polysaccharide (ASPS), a bioactive constituent extracted from Radix Astragali, may play an essential role for hematopoiesis. The objective of this study is to investigate the hematopoietic effects of ASPS in mouse models and explore underlying mechanism, which provides scientific basis for future applications. Methods: An irradiation-induced myelosuppressive mouse model and colony-forming unit (CFU) assays were used to determine the in vivo hematopoietic effects of ASPS. The anti-apoptotic effects of ASPS were also examined by using annexin V, caspase-3, and JC-1 assays with flow cytometry in HL-60 cells. Results: In the myelosuppressed mouse model, ASPS enhanced the recovery of platelet and red blood cell at day 21, and white blood cell at day 14. ASPS also promoted the CFU formation of all lineages (MK, GM, E, GEMM and F). Morphological study of bone marrow revealed that as compared to the control group, tri-lineage hematopoiesis was preserved in the ASPS- and TPO-treated group. Overall cellularity (MTC/area) of ASPS-treated group was similar to that of TPO. Furthermore, in-vitro study showed that 100 μg/ml of ASPS had maximum effect on CFU formation. ASPS also reduced apoptosis via inhibiting mitochondrial/Caspase-3 signal pathway. Conclusions: ASPS may promote hematopoiesis in irradiated myelosuppressive mice by enhancing hematopoietic proliferation and inhibiting apoptosis of hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

The CDK6 Inhibitor Palbociclib in Combination with Ruxolitinib Remarkably Improves Myelofibrosis in Murine Models

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF. We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF. We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells. We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib and ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination. We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF. Disclosures No relevant conflicts of interest to declare.

Recreation of t(7;12)(q36;p13) in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Generates a Useful Model for Studying Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

Efficient Elimination of Acute Myeloid Leukemia Cells through Inhibition of KDM4A in Combination with PARP Inhibition

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 & IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation > 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (>15% reduction) and increased apoptosis (>20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.

Surface PEGylation of MIL-101(Fe) nanoparticles for co-delivery of radioprotective agents

Providing protection from radiation damage is a great medical challenge. Metal-organic frameworks (MOFs) have high drug-loading capacities and excellent biocompatibility and are thus useful as drug-delivery systems. We fabricated a new drug carrier based on nanoscale MIL-101(Fe) to co-deliver two radioprotective agents by encapsulating WR-1065 (the active metabolite of amifostine) and glutathione (GSH). Consequently, the drug loading amounts of our drug carrier were 35.6 wt% and 6.5 wt% for WR-1065 and GSH, respectively. Moreover, radioprotective efficacy improved after PEGylation, which increased permeability and stability of these nanoparticles. In vitro studies showed that the MOF-based radioprotector had lower cytotoxicity compared with free WR-1065, and it increased cell viability by scavenging the accumulated reactive oxygen species and resisting DNA damage after irradiation. The MOF-based radioprotector—administered orally to mice—demonstrated efficacy at mitigating acute radiation syndrome; and its 30-d survival rate after lethal total body irradiation (TBI, 8 Gy) was 80%. The MOF-based radiation countermeasures led to significant improvements in peripheral blood cell count, hematopoietic stem and progenitor cells (HSPCs) frequency, and clonogenic function of hematopoietic progenitor cell. The ameliorated hematopoietic system injury in HSPCs is probably attributable to downregulation of BAX and BAK expression and upregulation of BCL-CL expression through the inhibition hematopoietic cell apoptosis. Therefore, the MOF-based radioprotectant has the potential to be used as a promising therapeutic candidate to attenuate TBI-induced hematopoietic injury.

PF660 TRANSCRIPTIONAL PROFILES OF PRIMARY CULTURES OF ERYTHROID AND MEGAKARYOCYTIC CELLS IN ESSENTIAL THROMBOCYTHEMIA UNCOVER A HIGH DEGREE OF GENE DEREGULATION IN THE MEGAKARYOCYTIC LINEAGE

Background: Essential Thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) characterized by megakaryocytic hyperplasia in the bone marrow and elevated number of platelets in the circulation. The driver mutations (JAK2V617F, MPLW515K/L, CALR Type I &II) in MPN have led to a better understanding of pathways involved in MPN, in particular the role of the JAK/STAT pathway. However, not much is known about the transcriptional programs in the pathogenesis of ET. Aims: We wanted to characterize the transcriptional signature of ET megakaryocytes to understand the molecular cues that drive the disease in the presence of different driver mutations. Methods: Progenitor cells were cultured from peripheral blood of 13 healthy donors (HD) and 16 untreated ET patients (JAK2V617F: n = 8; CALRType I: n = 4; triple-negative (TN-no JAK2, MPL, CALRmutations): n = 4). We obtained human erythroid (ERYs-proerythroblasts) and mature CD41+megakaryocytes (MKs) from each patient and healthy donor as described previously (Leberbauer, C. et al, 2005 and Salunkhe, V. et al, 2015) and RNA-seq was performed. Gene expression analysis data were selectively validated with qPCR for several targets. Results: Gene expression profiles from erythroid cultures and mature megakaryocytes (CD41+) from ET patients were grouped according to cell type (ERYs or MKs), type of driver mutations (JAK2, CALR) and compared with HD as controls. The expression profile of CALR mut ERYs was characterized by 60 significantly deregulated genes, 56 of which were upregulated (adjusted P-value 0.05) including non-erythroid targets (e.g. MPL, VWF, PF4, GP5, GP6). No major differences were identified between the transcriptional profile of HD ERY, JAK2V617F ERY or TN ERY. Similar comparisons were also made for the MKs. In a total of 4302 significantly deregulated genes (adjusted P-value 0.05) more than half (2348 genes) were uniquely up- or downregulated in the CALR mutant ET. Only 75 and 135 unique targets corresponded to JAK2V617F- and TN-MKs respectively and 925 deregulated genes were shared among the ET genetic groups. Principal component analysis (PCA) indicated that the MKs are more heterogeneous than ERYs and the JAK2V617F MK samples were the most variable of all. Gene ontology (GO) analysis showed that deregulated genes belong to the class of master regulators and hematopoietic cell fate, immune response, apoptosis, metabolic pathways and signal transduction. Some of these biological processes are shared by JAK2V617F and CALRmut-MKs (i.e. immune response). However, JAK-STAT and MAPK signaling pathways and apoptosis together form part of the unique transcriptional signature of CALRmut MKs. Summary/Conclusion: For the first time to our knowledge, paired cultures of committed erythroid cells and mature megakaryocytes from ET patients have been analyzed by RNA-seq. Comparison of ERY and MK transcriptional profiles show major differences between the two cell lineages, and between the driver mutations of CALR and JAK2. Our findings suggest that, in addition to JAK-STAT signaling, other mechanisms are involved in the transcriptional control in CALRmutated ET.

Excessive Reactive Iron Impairs Hematopoiesis by Affecting Both Immature Hematopoietic Cells and Stromal Cells.

Iron overload is the accumulation of excess iron in the body that may occur as a result of various genetic disorders or as a consequence of repeated blood transfusions. The surplus iron is then stored in the liver, pancreas, heart and other organs, which may lead to chronic liver disease or cirrhosis, diabetes and heart disease, respectively. In addition, excessive iron may impair hematopoiesis, although the mechanisms of this deleterious effect is not entirely known. In this study, we found that ferrous ammonium sulfate (FeAS), induced growth arrest and apoptosis in immature hematopoietic cells, which was mediated via reactive oxygen species (ROS) activation of p38MAPK and JNK pathways. In in vitro hematopoiesis derived from embryonic stem cells (ES cells), FeAS enhanced the development of dysplastic erythroblasts but inhibited their terminal differentiation; in contrast, it had little effect on the development of granulocytes, megakaryocytes, and B lymphocytes. In addition to its directs effects on hematopoietic cells, iron overload altered the expression of several adhesion molecules on stromal cells and impaired the cytokine production profile of these cells. Therefore, excessive iron would affect whole hematopoiesis by inflicting vicious effects on both immature hematopoietic cells and stromal cells.

Everolimus in combination with Imatinib overcomes resistance in Chronic myeloid leukaemia.

Although Imatinib and other tyrosine kinase inhibitors (TKIs) have excellent results, the appearance of resistance is a problem in chronic myeloid leukaemia (CML). PI3K/AKT/mTOR pathway is activated by BCR-ABL playing a crucial rule in CML. This study aimed to evaluate the therapeutic potential of Everolimus, in CML models sensitive and resistant to Imatinib. We used one CML cell line sensitive to Imatinib (K562) and two resistant (K562-RC and K56-RD). Cell lines were treated with Everolimus alone and in combination with Imatinib. Cell viability was analysed by resazurin assay. Cell death and cell cycle were analysed by flow cytometry. Additionally, we also studied peripheral blood samples obtained from 52 patients under TKI treatment. Everolimus reduced cell line viability in sensitive (IC50 = 20µM) and resistant models (K562-RC, IC50 = 25µM; K562-RD, IC50 = 30µM). This drug induced cell death by apoptosis and cell cycle arrest in G0/G1 phase. Everolimus also reduced cell viability by increasing apoptosis of haematopoietic stem cells (CD34+ cells) with low cytotoxicity to lymphocytes. Everolimus at 25µM increased apoptotic cells 18.7% in CD34+ cells and only 8% in lymphocytes. The response to Everolimus was influenced by TKI treatment, with a better response in samples from patients under 2nd and 3rd generation TKI and with less toxicity to lymphocytes. Our results reveal that Everolimus induce cell death in CML cells sensitive and resistant to Imatinib, with low cytotoxicity to normal cells, suggesting that Everolimus could be an alternative targeted therapeutic approach in CML patients, even in cases of Imatinib resistance.

Investigating the Role of C/Ebpδ-Deficiency in Radiation-Induced Bone Marrow Failure

Background: Bone marrow (BM) failure is a common side-effect of toxicity to the hematopoietic tissue both in the clinical setting as well as in case of total body irradiation (TBI) exposure in the event of nuclear terrorism or disasters. Particularly IR-induced myelosuppression is considered a significant risk factor for infections and increased risk for long-term hematopoietic dysfunction and myelodysplasia.Although a lot is known about the key regulatory proteins of steady-state hematopoiesis, not much is known about the regulatory factors involved in stress-induced hematopoiesis. Therefore delineating the mechanisms underlying the effects of IR stress-induced hematopoiesis is critical for the development of novel interventions with the potential to prevent or alleviate IR-induced BM injury/failure.The transcription factor CCAAT/enhancer-binding protein delta (Cebpd; C/EBPδ) plays an important role in the regulation of inflammatory and stress responses, and in the innate and adaptive immune responses. We have previously reported that Cebpd-knockout (KO) mice display increased neutropenia, thrombocytopenia and myelotoxicity in response to IR exposure, which correlated with increased apoptosis of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In the present study, we further investigated the underlying mechanisms of IR-induced bone marrow failure in the absence of C/EBPδ.Methods:Cebpd+/+and Cebpd-/- mice (C57BL/6J background) aged 3 months were exposed to 6 Gy TBI. Bone marrow mononuclear cells (BM-MNCs) were isolated from femurs and tibiae harvested at early time-points (1h, 4h and 24h) as well as 2 weeks post-irradiation. The presence of reactive oxygen species (ROS) was measured using MitoSOX and the extent of DNA damage was measured using an antibody specific to g-H2AX at the above time points. BM-MNCs were labelled with fluorophore-tagged antibodies and analyzed by flow cytometry to measure the absolute numbers of long term-HSCs, multipotent progenitors, common lymphoid progenitors and myeloid progenitors at 2 weeks post-6 Gy. The changes in cell cycle distribution in response to IR exposure will also be analyzed by flow cytometry using a DNA-binding dye in conjunction with Ki67.Results: Preliminary results reveal: (a) reduced numbers of HSCs, HPCs, common myeloid progenitors, myeloid-erythroid progenitors and granulocyte-monocyte progenitors and (b) increased accumulation of ROS and the DNA damage marker, γ-H2AX in HSCs and HPCs in Cebpd-/- mice compared to Cebpd+/+ mice at 2 weeks post-irradiation.These results suggest that C/EBPδ may play a protective role in radiation-induced bone marrow injury. Studies are underway to further examine changes in cell cycle, ROS and DNA damage in the various BM cell lineages post-TBI.(Funding support by NIGMS P20GM109005 & Department of Defense W81XWH-15-1-0489 is gratefully acknowledged) DisclosuresNo relevant conflicts of interest to declare.

CD34+ Derived Microparticles Regulate Apoptosis in Normal and Acute Myeloid Leukemia Cells

Introduction Microparticles (MPs) are small vesicles 100nm-1μm derived from the apoptotic or stimulated cells. The mechanism of their production is distinctive from exosomes or apoptotic bodies. MPs have been detected in the blood in many pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Our previous study showed that MPs originated also from CD34+ cells of umbilical cord blood, which is an alternative source for hemopoeitic stem cell transplantation. MPs considered as markers of cell activation, as well as apoptosis. Apoptosis is a complex interaction network regulated either through death receptor like FAS or through the internal pathway of Bcl2, Bax. The two pathways activate the executor of cell death program, caspases. The aim of this study is to elucidate the role of MPs in apoptosis of hemopoietic cells.Methods Umbilical cord blood units (UCB) were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. The HL60 promyelocytic leukemia cell line was cultured in RPMI (Life Technologies) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-strepromycin. CD34+ MPs were isolated from the plasma of UCBs after centrifugation and magnetic bead MACS purification (Miltenyi Biotec). The number of CD34+ MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation on lymphoprep (Fresenius) and were cultured for 3 and 6 days in the presence of CD34+ MPs. Viability assays were performed using 7-AAD in flow cytometry. In another set of experiments different numbers of CD34+ MPs were used in MNC cultures. RNA was extracted from MNC using Qiagen RNA extraction kit and reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) with random primers (Promega). RT-PCR was performed using Platinum Pfx polymerase (Invitrogen). The primers for FAS, BCL2, BAX, caspase 3, survivin and GAPDH genes were used. The PCR products were analysed in an agarose gel electrophoresis.Results Cell viability increased in UCB derived MNC (UCB-MNC) incubated with CD34+ MPs (800 /ml) after 3 day vs. 6 days of culture. The UCB-MNC viability was higher using 800/ml CD34+ MPs vs. 400/ml. In contrast, CD34+ MPs (800 /ml vs. 400/ml) did not affect the viability in one day MNC culture. Purified CD34+ MPs were applied to UCB-MNC cultures and by RT-PCR was shown increased expression of BCL2 gene as well as FAS and caspase-3 genes. The promyelocytc cell line HL60 has been used in order to analyze the effect of CD34+ MPs in leukemic cells. The expression of Bcl2 was decreased in HL60 cells co-incubated with CD34+ MPs. This result shows an opposite effect of CD34+ MPs in the apoptotic gene Bcl2 for the HL60 cells indicating that there are different mechanisms of MP function in various cell types.Conclusions In this study we have identified and monitored the time- and dose-dependent effect of CD34-derived microparticles in the viability of UCB mononuclear cells. Additionally, CD34+ MPs function is accosiated with the high expression of the pro- and anti-apoptotic Bcl2 and apoptotic FAS. In contrast CD34+ MPs decreases the expression of Bcl2 in the promyelocytic leukemia cell line HL60. Therefore the stem cell derived microparticles might serve as a potential regulator of apoptosis in normal and malignant hematopoietic cells. DisclosuresNo relevant conflicts of interest to declare.

Increased Age-Related B-Cells in Patients with Aplastic Anemia

Increased Age-Related B-cells in Patients with Aplastic AnemiaIntroduction:Aplastic anemia is a rare disease characterized by immune dysregulation. T cells in aplastic anemia are characterized by various intrinsic defects leading to increased IFN-g levels and Fas-mediated apoptosis of hematopoietic stem cells. We and others, have previously shown that the transcription factor T-bet is over-expressed in T cells from patients with aplastic anemia. Recently it was shown that a subpopulation of B cells also express T-bet; these cells are characterized as age-related B cells (ABCs) and express high levels of CD11c and CD19, they are CD21 negative and express T-bet. These T-bet+ ABCs are found increased in patients with autoimmune diseases (i.e. systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis). Stimulation of B cells with antigens, Toll-like receptors and IFN-g leads to the formation of ABCs, which in turn “talk” to the T cells and stimulate them. Stimulation of T cells leads to IFN-g production, and this IFN-g may lead to further induction of T-bet expressing B cells (ABCs).Specific aim: In this study we wanted to examine the expression of ABCs in patients with aplastic anemia. We isolated peripheral blood mononuclear cells (PBMCs) from patients with aplastic anemia (n=10) and eight healthy, age- matched controls. Written informed consent was obtained from all study subjects. Cells were stained with the surface markers CD11c, CD19 and CD21, and subsequently analyzed using flow cytometry. An anti-Tbet antibody was also used after cell permeabilization. The CD11c-high, CD19 positive, CD21 negative, T-bet positive population represent the ABCs.Results: Patients with aplastic anemia at presentation showed increased numbers of circulating ABCs compared to healthy controls (29,08±1,62% vs 4,06 ±0,4 % respectively, p<0.001).(Fig. 1). Patients who responded to treatment showed comparable levels of ABCs to those of healthy individuals (5,93±0,6% vs 4,06 ±0,4 % respectively). Non-responders had statistically significant increased levels of ABCs compared to healthy controls (p<0,001). Also, non-responders had ABCs levels similar to those of aplastic anemia patients at presentation (29,08±1,62% vs 31,7 ±2,97 % respectively, p=0,44).Conclusion: Although the number of patients analyzed is limited, our preliminary results suggest that aplastic anemia patients show expanded ABCs compared to age-matched control subjects, and these numbers are related to disease status. Further analysis of a larger pool of subjects is underway along with the examination of the specific transcription factor Bcl-6, that is implicated in T-bet expression in ABCs. These results will reveal the role of ABCs in the immune pathogenesis of aplastic anemia. DisclosuresKattamis:CELGENE: Consultancy, Honoraria; Vifor Pharma: Consultancy; Novartis: Consultancy, Honoraria; ApoPharma: Honoraria.

Gene alterations in monocytes are pathogenic factors for immunoglobulin a nephropathy by bioinformatics analysis of microarray data

BackgroundImmunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulopathy worldwide. The study aimed to provide potential molecular biomarkers for IgAN management.MethodsThe public gene expression profiling GSE58539 was utilized, which contained 17 monocytes samples (8 monocytes samples isolated from IgAN patients and 9 monocytes samples isolated from healthy blood donors). Firstly, differentially expressed genes (DEGs) between the two kinds of samples were identified by limma package. Afterwards, pathway enrichment analysis was implemented. Thereafter, protein-protein interaction (PPI) network was constructed and key nodes in PPI network were predicted using four network centrality analyses. Ultimately, gene functional interaction (FI) was constructed according to expressions in each sample, and then module network was extracted from FI network.ResultsA total of 678 DEGs were screened out, of these, 72 DEGs were identified as crucial nodes in PPI network that could well distinguish IgAN and healthy samples. In particular, IL6, TNF, IL1B, PRKACA and CCL20 were closely related to pathways such as hematopoietic cell lineage, apoptosis and Toll-like receptor (TLR) signaling pathway. Moreover, 12 genes in the FI network belonged to the 72 identified key nodes, such as CCL20, HDAC10, FPR2 and PRKACA, which were also key genes in 4 module networks.ConclusionsSeveral crucial genes were identified in monocytes of IgAN patients, such as IL6, TNF, IL1B, CCL20, PRKACA, FPR2 and HDAC10. These genes might co-involve in pathways such as TLR and apoptosis signaling during IgAN progression.

Selective bio-labeling and induced apoptosis of hematopoietic cancer cells using dual-functional polyethylenimine-caged platinum nanoclusters

In the past decades, platinum (Pt) is employed to clinical treatment of various cancers. However, for Pt-based drugs, especially Pt in +2 state [Pt (II)], such as cisplatin, number of drawbacks impede their anticancer efficiency including poor pharmacology, fast blood clearance, systemic toxicities causing from poor specificity and excretion of drug through kidneys. Herein, we developed dual-functional ultrafine polyethylenimine caged platinum nanoclusters (PEI-caged Pt NCs), which were utilized in biological imaging of the suspension cells system as fluorescent markers, and selective inhibition of hematopoietic malignancies as anticancer chemotherapeutics simultaneously. These zerovalent Pt NCs are capable to selectively enter into blood cancer cells (K562, BV173 cell lines) when compared to the peripheral blood mononucleated cells (PBMCs) from healthy donors, in addition, it can specifically induce pro-apoptotic protein expression (p53, PUMA, cleaved caspase) in hematopoietic cancer cells and promote cell apoptosis. Avoiding the adding of other fluorescent bio-markers, these Pt NCs showed great potential in diagnosis and treatment of hematopoietic system disease, such as acute myeloid leukemia, lymphoma, myeloma, etc.

Modeling ASXL1 mutation revealed impaired hematopoiesis caused by derepression of p16Ink4a through aberrant PRC1-mediated histone modification.

In spite of distinct clinical importance, the molecular mechanisms how Additional sex combs-like 1 (ASXL1) mutation contributes to the pathogenesis of premalignant conditions are largely unknown. Here, with newly generated knock-in mice, we investigated the biological effects of the mutant. Asxl1G643fs heterozygous (Asxl1G643fs/+) mice developed phenotypes recapitulating human low-risk myelodysplastic syndromes (MDS), and some of them developed MDS/myeloproliferative neoplasm-like disease after long latency. H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ hematopoietic stem cells (HSC), suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. The mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild-type ASXL1 protein. Restoration of HSC pool and amelioration of increased apoptosis in hematopoietic stem and progenitor cells wereobtained from Asxl1G643fs/+ mice heterozygous for p16Ink4a. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in low-risk MDS-like phenotypes in Asxl1G643fs/+ mice. This model provides a useful platform to unveil the molecular basis for hematological disorders induced by ASXL1 mutation and to develop therapeutic strategies for these patients.

Abstract 1874: The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis

Abstract Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild-type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. TP-3654 Treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and Ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth. We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with Ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and Ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas Ruxolitinib treatment did not significantly reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF. Citation Format: Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1874.