Treatment Of Myeloid Malignancies Research Articles

Targeting BIRC5 as a therapeutic approach to overcome ASXL1-associated decitabine resistance

Hypomethylating agents (HMAs) are widely employed in the treatment of myeloid malignancies. However, unresponsive or resistant to HMAs occurs in approximately 50 % of patients. ASXL1, one of the most commonly mutated genes across the full spectrum of myeloid malignancies, has been reported to predict a lower overall response rate to HMAs, suggesting an essential need to develop effective therapeutic strategies for the patients with HMA failure. Here, we investigated the impact of ASXL1 on cellular responsiveness to decitabine treatment. ASXL1 deficiency increased resistance to decitabine treatment in AML cell lines and mouse bone marrow cells. Transcriptome sequencing revealed significant alterations in genes regulating cell cycle, apoptosis, and histone modification in ASXL1 deficient cells that resistant to decitabine. BIRC5 was identified as a potential target for overcoming decitabine resistance in ASXL1 deficient cells. Furthermore, our experimental evidence demonstrated that the small-molecule inhibitor of BIRC5 (YM-155) synergistically sensitized ASXL1 deficient cells to decitabine treatment. This study sheds light on the molecular mechanisms underlying the ASXL1-associated HMA resistance and proposes a promising therapeutic strategy for improving treatment outcomes in affected individuals.

Innate Immune Mechanisms and Immunotherapy of Myeloid Malignancies.

Similar to other cancers, myeloid malignancies are thought to subvert the immune system during their development. This subversion occurs via both malignant cell-autonomous and non-autonomous mechanisms and involves manipulation of the innate and adaptive immune systems. Multiple strategies are being studied to rejuvenate, redirect, or re-enforce the immune system in order to fight off myeloid malignancies. So far, the most successful strategies include interferon treatment and antibody-based therapies, though chimeric antigen receptor (CAR) cells and immune checkpoint inhibitors are also promising therapies. In this review, we discuss the inherent immune mechanisms of defense against myeloid malignancies, currently-approved agents, and agents under investigation. Overall, we evaluate the efficacy and potential of immuno-oncology in the treatment of myeloid malignancies.

Stromal-Cells Downregulate MiR-23a-5p Levels in Myeloid Leukemic Cells to Activate Protective-Autophagy Against Chemotherapeutic Agents

Role of stromal micro-environment in drug resistance has been extensively reported for several cancers. We have demonstrated earlier that there is significant microenvironment mediated drug resistance (EMDR) to arsenic trioxide (ATO) in acute promyelocytic leukemia (APL) and that this was predominantly driven by up-regulation of the NF-kB pathway in the malignant cell (Ganesan S et al. Leukemia 2016). In our current study we have probed the molecular mechanism of EM-DR in further detail. Towards this we evaluated the potential role played by miRNA in EMDR to ATO in APL. Using NGS based small RNA sequencing we identified hsa-miR-23a-5p to be significantly downregulated (adjusted P value <0.01; figure 1a) in NB4 cells upon co-culture with HS-5 stromal cells. We noted that this miRNA is negatively regulated by NF-kB pathway and inversely correlated with NF-kB status in relapsed APL patients (n=8; matched samples). These results were consistent with our earlier reported observations that NF-kB pathway is dysregulated and enhances drug resistance to ATO. Further, we observed that low expression of miR-23a-5p observed in the relapsed patient's samples showed a trend towards an increased association with subsequent second relapse.We also observed miR-23a-5p mimics were able to restore the sensitivity of NB4 cells to ATO in the presence of stromal cells (figure 1b), while NB4/GFP-MAD cells (NF-kB inhibited cell line showing high expression of miR-23a-5p ) were not protected from ATO on stromal co-culture. Looking for the targets of miR-23a-5p , TargetScan analysis data demonstrated a clustering of targets involving toll like receptor genes (TLR) and autophagy pathway genes (TLRs - TLR2, TLR3, TLR4 and TLR5 and autophagy pathway genes - AMBRA1, ATG13, ATG9 and ATG2 ). Previously reported data clearly demonstrated the ability of TLR genes to activate autophagy (Delgado MA. et al. EMBO J 2008). Based on this data we evaluated the role of autophagy in EM-DR. We noted that there was an upregulation of autophagy pathway in leukemic cells upon co-culture with stroma (where miR-23a-5p was downregulated). Re-introducing miR-23a-5p in the leukemic cells targeted autophagy pathway genes which was validated through western blot (figure 1c). We also observed that inhibiting autophagy by chemical inhibitors (bafilomycin A1 and hydroxychloroquine) along with ATO was able to overcome EM-DR to ATO (figure 1d). The in-vivo efficacy of combining ATO along with hydroxychloroquine was further validated in an APL mouse model where we were able to demonstrate a significant decrease in tumor burden on day20 when compared to controls and significantly prolonged the survival of APL mice treated with the combination in comparison to either drug alone.Similar results were obtained with AML cells, where the stromal cells conferred a protective effect over U937 cells against cytarabine (AraC) and daunorubicin (Dnr). The downregulation of miR-23a-5p was also observed in U937 cells upon co-culture. We also observed that re-introducing miR-23a-5p in these cells restored sensitivity to AraC and Dnr. Our results thus illustrates a complex molecular cross-talk between stromal cells and leukemic cells in protecting against the cytotoxic effects of various chemotherapeutic agents in myeloid leukemia. This data along with that reported earlier by us illustrates multiple levels of regulation of the NF-kB pathway and resistance to chemotherapeutic drugs by stromal cell co-culture. Our work thus suggests that miR-23a-5p mimics or autophagy-inhibitors could potentially be effective adjuvants along with chemotherapy/ATO in the treatment of myeloid malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical Utility of Targeted Next-Generation Sequencing Assay to Detect Copy Number Variants Associated with Myelodysplastic Syndrome in Myeloid Malignancies

Copy number variants (CNVs) and gene mutations are important for diagnosis and treatment of myeloid malignancies. In a routine clinical setting, somatic gene mutations are detected by targeted next-generation sequencing (NGS) assay, but CNVs are commonly detected by conventional chromosome analysis and fluorescence in situ hybridization (FISH). The aim of this proof-of-principle study was to investigate the feasibility of using targeted NGS to simultaneously detect both somatic mutations and CNVs. Herein, we sequenced 406 consecutive patients with myeloid malignancies by targeted NGS and performed a head-to-head comparison with the results from a myelodysplastic syndrome (MDS) FISH and conventional chromosome analysis to detect CNVs. Among 91 patients with abnormal MDS FISH results, the targeted NGS revealed all 120 CNVs detected by MDS FISH (including -5/5q-, -7/7q-, +8, and 20q-) and 193 extra CNVs detected by conventional chromosome analysis. The targeted NGS achieved 100% concordance with the MDS FISH. The lower limit of detection of MDS CNVs by the targeted NGS was generally 5% variant allele fraction for DNA, based on the lowest percentages of abnormal cells detected by MDS FISH in this study. This proof-of-principle study demonstrated that the targeted NGS assay can simultaneously detect both MDS CNVs and somatic mutations, which can provide a more comprehensive genetic profiling for patients with myeloid malignancies using a single assay in a clinical setting.

Treatment of myeloid malignancies relapsing after allogeneic hematopoietic stem cell transplantation with venetoclax and hypomethylating agents\u2014a retrospective multicenter analysis on behalf of the German Cooperative Transplant Study Group

Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a great challenge. Aiming to evaluate the combination of venetoclax and hypomethylating agents (HMAClax) for the treatment of relapse of myeloid malignancies after alloHSCT, we retrospectively collected data from 32 patients treated at 11 German centers. Venetoclax was applied with azacitidine (n = 13) or decitabine (n = 19); 11 patients received DLI in addition. HMAClax was the first salvage therapy in 8 patients. The median number of cycles per patient was 2 (1–19). All but 1 patient had grade 3/4 neutropenia. Hospital admission for grade 3/4 infections was necessary in 23 patients (72%); 5 of these were fatal. In 30 evaluable patients, overall response rate (ORR) was 47% (14/30, 3 CR MRDneg, 5 CR, 2 CRi, 1 MLFS, 3 PR). ORR was 86% in first salvage patients versus 35% in later salvage patients (p = 0.03). In 6 patients with molecular relapse (MR), ORR was 67% versus 42% in patients with hematological relapse (HR) (n = 24, p = n.s.). After a median follow-up of 8.4 months, 25 patients (78%) had died and 7 were alive. Estimated median overall survival was 3.7 months. Median survival of patients with HMAClax for first versus later salvage therapy was 5.7 and 3.4 months (p = n.s.) and for patients with MR (not reached) compared to HR (3.4 months, p = 0.024). This retrospective case series shows that venetoclax is utilized in various different combinations, schedules, and doses. Toxicity is substantial and patients who receive venetoclax/HMA combinations for MR or as first salvage therapy derive the greatest benefit.

Energy metabolism and drug response in myeloid leukaemic stem cells.

Despite significant advances in the treatment of myeloid malignancies, many patients become resistant to therapy and ultimately succumb to their disease. Accumulating evidence over the past several years has suggested that the inadequacy of many leukaemia therapies results from their failure to target the leukaemic stem cell (LSC). For this reason, the LSC population currently represents the most critical target in the treatment of myeloid malignancies. However, while LSCs are ideal targets in the treatment of these diseases, they are also the most difficult population to target. This is due to both their heterogeneity within the LSC population, and also their phenotypic similarities with normal haematopoietic stem cells. This review will highlight the current landscape surrounding LSC biology in myeloid malignancies, with a focus on altered energy metabolism, and how that knowledge is being translated into clinical advances for the treatment of chronic and acute myeloid leukaemia and myelodysplastic syndromes.

The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies

Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies.

Abstract DDT02-04: BAY 2402234: A novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of myeloid malignancies

Abstract Acute myeloid leukemia (AML), the most common acute leukemia in adults, is an aggressive hematologic malignancy resulting in bone marrow failure with a poor outcome; overall survival is approximately 25% at five years. Treatment options, in particular for the elderly population, are limited. Induction chemotherapy of cytarabine and an anthracycline (7+3) remains unchanged standard of care since its introduction in the early 1970s and there is a high medical need for new therapies (Yates et al. Cancer Chemother Rep 1973). DHODH is a key enzyme in the de novo pyrimidine synthesis converting dihydroorotate to orotate. Using a HOXA9 driven phenotypic screen to overcome differentiation arrest in myeloid cells, we have recently identified DHODH as a surprising novel target to overcome differentiation blockade in AML (Sykes et al. Cell 2016). Differentiation therapy already showed its enormous clinical benefit potential in the small subset of patients diagnosed with acute promyelocytic leukemia (APL) following treatment with all-trans retinoic acid with five-year survival exceeding 85% and should be considered the ultimate therapeutic goal for all AML subsets (Lo-Coco et al. NEJM 2013). Here, we disclose for the first time the structure and functional characterization of the novel DHODH inhibitor BAY 2402234. BAY 2402234 is a selective low-nanomolar inhibitor of human DHODH enzymatic activity. In vitro, it potently inhibits proliferation of AML cell lines in the sub-nanomolar to low-nanomolar range. BAY 2402234 induces differentiation of AML cell lines also in a sub-nanomolar to low-nanomolar range, demonstrating the anticipated mode of action in cellular mechanistic assays. In vivo, BAY 2402234 exhibits strong in vivo anti-tumor efficacy in monotherapy in several subcutaneous and disseminated AML xenografts as well as AML patient-derived xenograft (PDX) models. Target engagement of the novel DHODH inhibitor BAY 2402234 can be observed by increase of tumoral and plasma dihydroorotate levels after treatment with the inhibitor. Consistent with the in vitro data BAY 2402234 induces AML differentiation in vivo as detected by upregulation of differentiation cell surface markers in xenograft and PDX models after treatment with the inhibitor. Furthermore, differentiation-associated transcriptomic changes were evident following a single administration of BAY 2402234 in vivo. The start of clinical investigations of BAY 2402234 is planned for early 2018. Citation Format: Andreas Janzer, Stefan Gradl, Sven Christian, Katja Zimmermann, Claudia Merz, Hanna Meyer, Timo Stellfeld, Judith Guenther, Detlef Stoeckigt, Henrik Seidel, Pascale Lejeune, Michael Bruening, Ashley Eheim, Thomas Mueller, Ralf Lesche, Martin Michels, Andrea Haegebarth, Marcus Bauser, Sherif El Sheikh, Steven Ferrara, David Sykes, David Scadden. BAY 2402234: A novel, selective dihydroorotate dehydrogenase (DHODH) inhibitor for the treatment of myeloid malignancies [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 DDT02-04.

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34+CD38+CD33+ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3+ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.

Isocitrate dehydrogenase (IDH) inhibition as treatment of myeloid malignancies: Progress and future directions.

Isocitrate dehydrogenase (IDH) is an essential metabolic enzyme. Over the last two decades, there has been a growing focus on the metabolic derangements that occur with IDH1 and IDH2 mutations. The altered IDH protein leads to accumulation of 2-hydroxyglutarate (2-HG), a metabolite with oncogenic activity via epigenetic mechanisms. The advent of IDH inhibitors has engendered hope in novel and targeted therapies in IDH1/2 mutant myeloid malignancies. We here summarize the basic physiology of IDH, the metabolic and oncogenic consequences of mutant IDH1/2, and the clinical significance of IDH inhibition in hematologic malignancies. We also discuss completed and ongoing clinical trials focusing on the inhibition of IDH proteins, which have demonstrated preliminary indications of efficacy. The promise of IDH inhibition is now being further investigated as a novel therapeutic approach for AML and other myeloid malignancies.

Survival Benefit of Intravenous Busulfan (120 mg/M2) and Fludarabine Myeloablative Regimen for Treatment of Myeloid Malignancies

Background: Intravenous fludarabine and busulfan 130 mg/m2 (Flu/Bu) conditioning regimen has induced the long term survival with a low treatment related mortality rate. However, there have been few reports on long term survival of patients undergoing allo-HSCT with intravenous Flu/Bu regimen. Therefore, we conducted a retrospective study of 42 patients diagnosed with myeloid malignancies received allo-HSCT with intravenous fludarabine and busulfan (120 mg/m2) regimen between 2006 and 2015 at Ramathibodi hospital. The aim of our study was to observe the long term survival and the complication after transplantation. Findings: Thirty- four, three and five patients were AML, MDS and CML-CP respectively. With a median followup of 95 months, 1- year EFS and 8- year EFS were 82 and 70% respectively. Overall survival (OS) rate at 1 and 8 years were equally observed in 88%. Patient younger than age 45 years had significantly longer OS than patients aged 45 years and older (96 vs 70%, p=0.019). Eight- year OS in AML, MDS and CML were 88, 67 and 100% respectively. Acute and chronic graft versus host disease were found in 29 and 46.3% of 41 evaluable patients respectively. Whereas the rates of sinusoidal obstruction syndrome, sepsis, CMV reactivation, cyclosporine and tacrolimus induced thrombotic microangiopathy were 2, 10, 12, 5 and 2% respectively. Non- relapse mortality rate at day +100, 1 and 8 year were only 9.5, 13.8 and 13.8% respectively. There was no neurological toxicity, severe mucositis, secondary malignancy or therapy related MDS syndrome in this study. Conclusion: Allogeneic hematopoietic stem cell transplantation with intravenous fludarabine and busulfan at the dose of 120 mg/m2 was well tolerated and demonstrated impressive treatment outcomes in young adult patients diagnosed with myeloid malignancies.

5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

Abstract 677: Transient exposure to decitabine results in sustained cell growth inhibition and long term DNA demethylation at specific loci.

Abstract Gene silencing mediated by aberrant promoter DNA hypermethylation is one of the key features of cancer. Although such modification is heritable, its dynamic nature and reversibility through pharmacological interventions make it an attractive therapeutic target. Over the past few decades, various DNA methyltransferase inhibitors have been developed with the goal of reactivating aberrantly silenced genes. Two FDA-approved demthylating agents, decitabine and azacitidine are efficacious for the treatment of myeloid malignancies (MDS). It has been shown that transient exposure of tumor cells to low dose decitabine and azacitidine results in reduced tumorigenecity. However, the mechanism underlying clinical efficacies of these DNA methyltransferase inhibitors remains unclear. Here we investigate the long-term effect (up to 10 weeks) on population doubling time and the returning of DNA methylation (rebound methylation) after transient exposure to decitabine in three cancer cell lines, HCT116, T24 and HL-60. We show that transient exposure results in inhibition of cell growth and reduction of their colony formation capacity (up to 42 days). Furthermore, we also show that the rebound methylation occurs at different rates depending on the region and the type of genes. The discrepancy in population doubling time between the vehicle and decitabine treated cells disappears when rebound methylation fully restores, suggesting the presence of a correlation between cell doubling and rebound methylation. The majority of probes, which exhibit fast rebound methylation, are located in gene bodies. We show a positive correlation between gene body methylation and expression. However, there is a small group of fast rebound probes located at the promoters. Many of them belong to cancer-testis antigens, which get reactivated and quickly re-silenced. On the other hand, the majority of probes, which exhibit slow rebound methylation are located at promoters and show an inverse correlation between methylation and expression. The presence of a nucleosome-depleted region and enrichments of active histone marks, H3K4me3 and H2A.Z are observed at the promoters of those genes up to 42 days after drug treatment. Interesting, this group is enriched for genes down-regulated in colon adenocarcinoma when compared to normal colon (p=3.3e−6). Taken together, the sustained reactivation of those genes may be the driving force in reducing tumorigenecity of cancer cells. Our study elucidates the mechanism of decitabine, thus advancing our understanding of epigenetic therapy. Citation Format: Gangning Liang, Han Han, Daniel D. De Carvalho, Xiaojing Yang, Peter A. Jones. Transient exposure to decitabine results in sustained cell growth inhibition and long term DNA demethylation at specific loci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 677. doi:10.1158/1538-7445.AM2013-677 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

STAT signaling in the pathogenesis and treatment of myeloid malignancies

STAT transcription factors play a critical role in mediating the effects of cytokines on myeloid cells. As STAT target genes control key processes such as survival, proliferation and self-renewal, it is not surprising that constitutive activation of STATs, particularly STAT3 and STAT5, are common events in many myeloid tumors. STATs are activated both by mutant tyrosine kinases as well as other pathogenic events, and continued activation of STATs is common in the setting of resistance to kinase inhibitors. Thus, the targeting of STATs, alone or in combination with other drugs, will likely have increasing importance for cancer therapy.

Abstract 3570: SB1518, a novel JAK2/FLT3 inhibitor for the treatment of myeloid malignancies

Abstract Introduction: Janus kinase 2 (JAK2) and fms-related tyrosine kinase 3 (FLT3) are important tyrosine kinases in hematopoietic progenitor cell differentiation and proliferation. Aberrant signaling of JAK2 or FLT3 is implicated in various myeloid malignancies. SB1518, a new chemical entity, is an inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) as well as FLT3 (IC50 = 22 nM) and its mutant FLT3D835Y (IC50 = 6 nM). It is currently being evaluated in Phase I and II clinical trials for advanced hematologic malignancies and is showing promising therapeutic effects. Here we present a pharmacological evaluation of SB1518 in in vitro and in vivo models of myeloid disease that provide a rationale for its selection for clinical development. Methods: The inhibitory effect of SB1518 on JAK2, FLT3 and STAT signaling was evaluated by Western-blot analysis in cell lines with different JAK2/FLT3 mutation status. Cell proliferation assays using the CellTiterGlo® assay were performed on a panel of different cancer cell lines and primary cells from patients. Selected cells were treated with SB1518 to determine effects on apoptosis (Caspase 3/7 activation) and cell cycle (propidium iodide stain). SET-2 cells (a megakaryoblastic cell line derived from an ET-patient bearing JAK2V617F) and MOLM-13 cells (an AML cell line bearing FLT3-ITD) were used to establish subcutaneous tumors in nude mice to test the anti-tumor efficacy of SB1518 after oral daily dosing. Results: SB1518 inhibits phosphorylation of JAK2 at Y221, FLT3 and of STAT proteins and shows anti-proliferative activity in cell lines driven by mutant or wt JAK2 or FLT3-ITD (IC50 range of 47-348 nM), leading to cell cycle arrest (in G0/1) and induction of apoptosis. Cells bearing JAK2 or FLT-3 mutations or genetic alterations leading to high expression of JAK2, are the most sensitive to the anti-proliferative effects of SB1518, as are primary cells isolated from PV or AML patients. In these cells SB1518 induces cell cycle arrest and apoptosis. SB1518 has favorable physicochemical properties and pharmacokinetics allowing oral dosing. It significantly blocks tumor growth in a subcutaneous model of megakaryoblastic AML (SET-2 cells, TGI = 61% at 150 mg/kg b.i.d.). In a model of late stage, aggressive AML (MOLM-13), SB1518 not only blocked tumor growth (TGI = 81%, at 150 mg/kg b.i.d.), but also significantly reduced lung metastasis. In both models SB1518 was very well tolerated and did not induce cytopenia. Conclusions: SB1518, a novel, orally bio-available small molecule inhibitor of JAK2/FLT3, shows promising activities in various hematological cancer cell lines as well as primary cells from patients. It is efficacious in mouse xenograft models of FLT-3 dependent MOLM-13 cells and JAK2-dependent SET-2 cells. SB1518 is currently undergoing evaluation in Phase 2 clinical trials in myelofibrosis (post-PV, post-ET and primary) and has shown encouraging clinical activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2011-3570

The interaction between taxoids and serine/threonine protein phosphatase activities during taxan-induced apoptosis of HL 60 leukemic cells

Paclitaxel and docetaxel (taxoids) are chemotherapy agents whose mode of action is through an effect on cellular microtubules. Several studies have investigated their potential in the treatment of myeloid malignancies. The aim of our study was to investigate the potential role of the serine/threonine protein phosphatase system in docetaxel/paclitaxel induced cytotoxicity on HL 60 cells. The IC50 dose of paclitaxel and docetaxel were found as 20 and 5 nM respectively using trypan blue dye exclusion and XTT assays. Treating HL 60 cells with docetaxel and paclitaxel resulted in dose and time dependent cytotoxicity. Docetaxel induced the decrease in the activity of protein phosphatase 1 (PP1) and increase in the activity of PP2 subgroups, while paclitaxel induced the increase in the activity of PP1 and decrease in the activity of PP2 subgroups. Potential use of specific protein phosphatase inhibitors or activators in combination with taxoids will open new windows in the treatment of myeloid leukemias.

Reply to Valproic Acid for the Treatment of Myeloid Malignancies

Reply to Valproic Acid for the Treatment of Myeloid Malignancies

156: Treatment of Myeloid Malignancies in Elderly Patients with Fludarabine and Targeted Busulfan (t-Bu) and Allogeneic Hematopoietic Cell Transplantation (HCT)

156: Treatment of Myeloid Malignancies in Elderly Patients with Fludarabine and Targeted Busulfan (t-Bu) and Allogeneic Hematopoietic Cell Transplantation (HCT)

MicroRNA-155 Promotes Myeloid Proliferation and Is Overexpressed in Acute Myeloid Leukemia.

Recent discoveries have implicated microRNAs, which are small 22–24 nucleotide long RNA molecules, as important regulators of many cellular processes, including differentiation and development. The microRNA-155 (miR-155) is known to be overexpressed in subsets of B-cell neoplasms and is thought to be important in the activation and function of B-lymphocytes. Here, we show that miR-155 is signficantly overexpressed in human acute myeloid leukemia and that its overexpression is most consistently seen in acute myelomonocytic leukemia. These findings led us to investigate the effects of overexpression of miR-155 in hematopoietic cells. By utilizing retroviral infection and transfer of murine bone marrow, we introduced miR-155 overexpressing hematopoietic stem cells into lethally irradiated recipient mice. Following reconstitution of hematopoietic organs, the mice demonstrated a profound myeloproliferative condition in the bone marrow characterized by replacement of the marrow by proliferating and dysplastic myelomonocytic cells. In addition, extramedullary hematopoiesis was observed in the spleen and examination of the peripheral blood revealed anemia and thrombocytopenia. To begin to explore the mechanisms whereby miR-155 overexpression results in this myeloproliferative phenotype, we utilized computational methods to identify targets predicted to be regulated by miR-155. This revealed several genes that have previously been implicated in myeloid development and neoplasia, which were confirmed to be targets of miR-155 by reverse-transcription/quantitative polymerase chain reaction (RT/QPCR) and by downregulation of luciferase protein upon fusion of the luc gene with the respective 3′ untranslated regions. These studies show a hitherto uncharacterized role of miR-155 in myeloid development and proliferation. Importantly, these findings lay the groundwork for understanding the complex regulatory networks underlying myeloid development in the context of microRNAs, and may point to new therapeutic opportunities in the treatment of myeloid malignancies.

Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression

The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.