Treatment Of Acute Myeloid Leukemia Cells Research Articles

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

AbstractThe transcriptional repressor BCL11A is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase Trib1 in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells in vitro and in vivo. Chromatin immunoprecipitation sequencing analysis showed that, upon DNA binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components HDAC and LSD1 reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor pracinostat and the LSD1 inhibitor GSK2879552 resulted in growth inhibition in vitro and in vivo. High BCL11A expression is associated with worse prognosis in humans with AML. Blocking of BCL11A expression upregulates the expression of PU.1 target genes and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

Differentiation of acute myeloid leukemia (AML) cells with ATRA reduces 18F-FDG uptake and increases sensitivity towards ABT-737-induced apoptosis

Acute myeloid leukemia (AML) is a malignant disease of the bone marrow, comprising various subtypes. We have investigated seven different AML cell lines that showed different sensitivities toward the inducer of apoptosis ABT-737, with IC50 concentrations ranging from 9.9 nM to 1.8 µM. Besides, the AML cell lines revealed distinct differences in 18F-FDG uptake ranging from 4.1 to 11.0%. Moreover, the Pearson coefficient (0.363) suggests a moderate correlation between 18F-FDG uptake and the IC50 values of ABT-737. Differentiation of the AML cell lines NB-4 and AML-193 with all-trans-retinoic-acid (ATRA) induced a significant increase in sensitivity towards ABT-737 along with a reduced uptake of 18F-FDG. Therefore, 18F-FDG uptake could be predictive on sensitivity to treatment with ABT-737. Furthermore, because differentiation treatment of AML cells using ATRA reduced 18F-FDG uptake and increased sensitivity towards ABT-737, a combined treatment regimen with ATRA and ABT-737 might be a promising therapeutic option in the future.

DNA Methyltransferase Inhibitors Promote Homologous Recombination Deficiency through Induction of Immune Signaling, Sensitizing Acute Myeloid Leukemia Cells to PARP Inhibitors

Acute myeloid leukemia (AML) patients unfit for intensive chemotherapy are treated with DNA methyltransferase inhibitors (DNMTis). However, while many AML patients respond to DNMTis, responses are not durable. We previously reporteda novel treatment strategy for AML that combines DNMTis with poly (ADP-ribose) polymerase inhibitors (PARPis), drugs classically used to treat breast and ovarian cancer patients with BRCA mutations and homologous recombination defects (HRD) (Faraoni and Graziani, 2018). We found that combining low doses of the potent PARP-trapping PARPi talazoparib with DNMTis increases PARP trapping and cytotoxicityin vitroand increases therapeutic efficacy in vivo (Muvarak et al, 2016). We have nowidentified a novel mechanism through which DNMTis may sensitize BRCA-proficient AML cells to PARPis. This mechanism is tied to the capacity of these drugs to reprogram cancer signaling networks, including altering DNA repair pathways (Tsai et al, 2012). In studies in AML cell lines (N=6) and peripheral blood mononuclear cells (PBMCs) from AML patients (N=4), we now show that treatment with the DNMTi decitabine (DAC) at a low concentration (10nM) can directly induce HRD, by significantly (p<0.01) down-regulating key genes central to HR activity, including multiple genes in the Fanconi anemia (FA) pathway, as a mechanism for enhanced PARPi sensitivity. How do DNMTis downregulate HR gene expression? We show for the first time that immune signaling is linked to induction of HRD. We have previously shown that DNMTis activate innate immune pathways involving interferon (IFN) □ and tumor necrosis factor (TNF) □, a phenomenon known as viral mimicry (Chiappinelli et al, 2015).First, The Cancer Genome Atlas (TCGA) AML data sets show an inverse correlation between type 1 interferon (IFN)/pro-inflammatory response and HR-related genes. Second, we verified in BRCA-proficient AML cell lines (N=6) that immune signaling by exogenous TNF□□or IFN□□treatment decreases HR gene expression and activity by more than two-fold for the majority of genes tested (p<0.0001). Third, treatment of AML cells with IFN□and the signal transducer and activator of transcription (STAT) 1/3 inhibitor ruxolitinib can rescue DAC-induced HRD. Importantly, we identified a common immune signaling pathway induced by both DNMTis and PARPis. PARPis have also been shown to activate type 1 IFN pathways via induction of cytoplasmic double-stranded DNA sensing through signaling of the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes(cGAS-STING) pathway. We now find that inhibition of STING with inhibitor H-151 (500nM) not only rescues immune signaling induced by PARPi, but also by DAC and PARPi combination treatment. Moreover, the STING inhibitor also rescues DAC- and/or PARPi-induced HRD. These data suggest that STING may be a central signaling hub linked to HRD and also suggest ways in which epigenetic therapy, inhibitors of DNA damage response proteins, and targeted immune therapy can synergize to treat AML. Disclosures Baer: Takeda: Research Funding; Incyte: Research Funding; Kite: Research Funding; Forma: Research Funding; AI Therapeutics: Research Funding; Abbvie: Research Funding; Astellas: Research Funding.

Selinexor in Combination with Induction and Consolidation Therapy in Older Adults with AML Is Highly Active

Background: Acute myeloid leukemia (AML) is an aggressive myeloid cancer of the hematopoietic system that primarily affects older adults and is characterized by therapy resistance and dismal outcomes. Novel approaches to treat AML are desperately needed. Selinexor is a small-molecule inhibitor of the nuclear export protein XPO1. Treatment of AML cells with selinexor was shown to sensitize them to chemotherapy in part by blocking the nuclear export of topoisomerase II, resulting in increased DNA strand breaks when an anthracycline is present. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in the relapsed setting. This abstract reports on the initial results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML 60 years of age or older. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized between 7+3 or 7+3+selinexor induction. Responding patients could then go on to high dose cytarabine consolidation with or without selinexor as per their initial treatment assignment. Patients in the selinexor arm who completed all planned consolidation could then move to maintenance therapy with selinexor alone. During induction cytarabine was dosed at 100 mg/m2 by continuous infusion for 7 days and daunorubicin was dosed at 60 mg/m2 on days 1-3. In consolidation cytarabine was dosed at 1.5 gm/m2 given Q12 hours on days 1, 3 and 5. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days in maintenance. Optional blood samples were collected just prior to and at several time points after selinexor during induction. Mononuclear cells were isolated from these samples, lysed and lysates evaluated for protein pathway drug target activation mapping by reverse phase phosphoprotein array (RPPA). Results: Thirteen patients have been enrolled to date with 12 available for evaluation. Of the 12 evaluable patients 6 were randomized to each arm. Baseline demographics are listed in Table 1. Overall the combination was well tolerated with no patients discontinuing selinexor secondary to treatment related adverse events. In the standard arm 3 of 6 patients achieved a complete remission. Of the 3 responders 1 has relapsed, 1 has gone on to transplant and one has completed consolidation chemotherapy. In the selinexor arm 5 of 6 patients achieved a complete remission and 1 patient achieved a morphologic leukemia free state. All patients given selinexor achieved a response. Of the 5 responders 3 have gone on to transplant, 1 is on maintenance therapy (completed 14 cycles) and one is completing consolidation therapy. In the standard arm no patients died during induction and 3 of 6 patients have died from progressive disease. In the selinexor arm 1 patient died during induction and none of the remaining patients have progressed (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. The patient on long term maintenance selinexor is tolerating it without significant AEs to date. Blood samples were available from 3 patients from the selinexor arm. RPPA based protein pathway activation mapping analysis of PBMCs revealed a significant increase in phosphorylation of AMPK, BAD and LC3B at one-hour post treatment that returned to baseline by 2 hours suggesting a transient increase in these pathways that was subsequently suppressed (Figure 2). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older de novo AML patients. Selinexor may increase response by modulation of AMPK, autophagy and BAD phosphorylation. Enrollment is ongoing. Disclosures Pardee: Spherix Intellectual Property: Research Funding; CBM Bipharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Celgene: Speakers Bureau; Pharmacyclics/Janssen: Speakers Bureau; Karyopharm: Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding. Manuel:Novartis: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Powell:Pfizer: Consultancy, Research Funding; Rafael Pharmaceuticals: Consultancy, Research Funding; Novartis: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Research Funding, Speakers Bureau; Janssen: Research Funding. OffLabel Disclosure: Selinexor is not approved for the treatment of AML

Camel Whey Protein Disrupts the Cross-Talk Between PI3K and BCL-2 Signals and Mediates Apoptosis in Primary Acute Myeloid Leukemia Cells

In the present study, we investigated the impact of camel whey protein (CWP) on the survival of primary acute myeloid leukemia (AML) cells that were isolated from 20 patients diagnosed with AML. We found that CWP induced apoptosis in the primary AML cells without affecting the normal PBMCs that were isolated from healthy individuals, as determined by PI/annexin V double staining followed by flow-cytometry analysis. Furthermore, we demonstrated that these primary AML cells exhibited aberrant phosphorylation of AKT, mTOR and STAT3. Treatment of AML cells with CWP mediated significant reduction in the phosphorylation of AKT, mTOR and STAT3. Additionally, we demonstrated that blockade of PI3K/AKT signaling pathway by wortmannin (WM) impaired the expression of Bcl-2 and BclXL in the primary AML cells, suggesting an essential cross-talk between PI3K and Bcl-2 that maintains the survival of AML cells. In this context, treatment of AML cells with CWP disrupted the PI3K/Bcl-2 cross-talk; significantly downregulated the expression of anti-apoptotic Bcl-2 family members Bcl-2 and BclXL; markedly upregulated the expression of the pro-apoptotic Bcl-2 family members Bak and Bax; and subsequently sensitized tumor cells to growth arrest. Our data revealed the therapeutic potential of CWP and the underlying mechanisms against leukemia.

Inhibition of ATR Potentiates Acute Myeloid Leukemia Cells to the RNA Pol I Transcription Inhibitor CX-5461

Inhibition of ATR Potentiates Acute Myeloid Leukemia Cells to the RNA Pol I Transcription Inhibitor CX-5461

Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/β-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents.Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments.Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively.Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3α, (Ser21) GSK-3α, total GSK-3β, and (Ser 21) GSK-3β; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin.Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes. DisclosuresBonifacio:Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Amgen: Consultancy.

Inhibition of CHK1 Enhances Cell Death Induced By the Bcl-2-Selective Inhibitor ABT-199 in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) remains a challenging disease to treat in both pediatric and adult populations. Resistance to cytarabine (ara-C) and anthracycline [e.g., daunorubicin (DNR)]-based chemotherapy is a major cause of treatment failure in this disease. Therefore, more effective therapies are urgently needed to improve treatment outcome of AML patients.Anti-apoptotic Bcl-2 family proteins play key roles in the apoptosis pathway. Overexpression of these proteins is associated with chemoresistance and poor clinical outcome. Thus, much attention has been focused on inhibition of the anti-apoptotic Bcl-2 family proteins for the treatment of various malignancies. ABT-199 is a selective Bcl-2 inhibitor that has demonstrated promising results in CLL, as well as other malignancies including AML. We previously demonstrated that ABT-199 has a wide range of activity in AML cells. In addition, we have also identified that the anti-apoptotic protein Mcl-1, in conjunction with the pro-apoptotic protein Bim, is a key player in resistance to ABT-199 in AML cells. Thus, combining ABT-199 with agents that downregulate Mcl-1 could overcome the intrinsic resistance to ABT-199.Checkpoint kinase 1 (CHK1) is a protein kinase which plays a central role in the DNA damage response (DDR). The DDR represents a complex network of multiple signaling pathways involving cell cycle checkpoints, DNA repair, transcriptional programs, and apoptosis, through which cells maintain genomic integrity following various endogenous or environmental stresses. Inhibition of CHK1 has been demonstrated to induce DNA damage. We and others have also demonstrated that DNA damage results in downregulation of Mcl-1. Thus, it is conceivable that targeting CHK1 may enhance the cytotoxic effects of ABT-199 on AML cells through downregulation of Mcl-1.In this study, we investigated the combination of LY2603618, a CHK1-selective inhibitor, and the Bcl-2 inhibitor ABT-199 in AML cell lines and primary patient samples. We demonstrated that LY2603618 inhibited proliferation of AML cell lines (n=11) and diagnostic blasts (n=26). Interestingly, all 11 AML cell lines and 23 out of the 26 primary AML patient samples tested showed a LY2603618 IC50 lower than the Cmax of LY2603618 (~9 µM) determined in Phase I clinical studies. Annexin V and propidium iodide (PI) staining and flow cytometry analyses revealed that LY2603618 induced Bak-dependent apoptosis in AML cells. As expected, treatment of AML cells with LY2603618 resulted in abolishment of G2 cell cycle check point and DNA double strand breaks (DSBs), which could be, at least partially, blocked by a CDK inhibitor, roscovitine. LY2603618 treatment also resulted in downregulation of Mcl-1, which coincided with the initiation of apoptosis. Overexpression of Mcl-1 in AML cells significantly attenuated apoptosis induced by LY2603618, confirming the critical role of Mcl-1 in apoptosis induced by the agent. Consistent with our hypothesis, simultaneous combination of LY2603618 and ABT-199 resulted in synergistic induction of apoptosis in both AML cell lines and primary patient samples.Our results demonstrate that LY2603618 synergizes with ABT-199 in AML cells. Our findings provide new insights into overcoming the mechanism of ABT-199 resistance in AML cells and support the clinical development of the combination of ABT-199 and CHK1 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

SGN-CD33A in Combination with Hypomethylating Agents Is Highly Efficacious in Preclinical Models of AML

Acute myeloid leukemia (AML) remains a therapeutic challenge. Long-term survival rates, in particular for older AML patients, remain poor, highlighting the need for improved and well-tolerated treatment options. AML patients who are unfit for high dose chemotherapy in the US are often prescribed hypomethylating agents (azacitidine or decitabine) although the efficacy of these agents in this population was modest (Estey, Leukemia 2013).SGN-CD33A is a CD33-directed antibody drug conjugate (ADC) that is comprised of a cysteine-engineered anti-CD33 antibody, a cleavable dipeptide linker that is highly stable in circulation, and a PBD dimer that binds DNA with high intrinsic affinity. The ADC is active as a single agent in preclinical models of AML that are characteristically resistant to chemotherapy (multi-drug-resistant, MDR-positive) (Sutherland et al. Blood 2013). In the present study, we tested the activity of SGN-CD33A in combination with hypomethylating agents (HMA), azacitidine or decitabine. We hypothesized that the combination of SGN-CD33A with an HMA will have greater impact on the DNA repair pathway in leukemic cells, furthering the processes of apoptosis and cell death. MDR-positive AML cells were treated for 96 hours with SGN-CD33A and each of the HMAs alone and in combination, evaluating both simultaneous as well as sequential administration. Enhanced tumor cell killing was observed when AML cells were treated concomitantly with the combination of SGN-CD33A and an HMA or pre-treated with HMAs prior to the addition of SGN-CD33A.Superior anti-leukemic activity of subtherapeutic doses of SGN-CD33A in combination with HMAs was observed in mouse xenograft models generated with MDR-positive AML cell lines. In the difficult to treat HEL9217 model, decitabine or a single dose of 200 mcg/kg SGN-CD33A delayed tumor growth, while significant reductions in tumor growth were observed for the combination treatment (p=0.0001). Improved antitumor activity in this model was also observed for SGN-CD33A in combination with azacitidine. SGN-CD33A in combination with decitabine significantly reduced tumor burden compared to either agent alone in the TF1-α AML model (p=0.0002). Similarly, sequenced dosing of azacitidine followed by a subtherapeutic dose of 30 mcg/kg SGN-CD33A in mice bearing KG-1 xenografts delivered greater antitumor activity compared to the individual agents (p=0.0001).To investigate the mechanism underlying the enhancement in antileukemic activity, we looked at the impact of HMAs alone and in combination with SGN-CD33A on myeloid marker expression, PBD drug release, and impact on the DNA damage and apoptotic pathways. In AML cell lines that did not show improved cytotoxic activity with the combination of SGN-CD33A and HMA, HMA treatment appeared to have no positive effect on the cell surface levels of CD33. However in responsive cell lines such as TF1-α, HMA treatment resulted in time- and dose-dependent increases in the levels of CD33. Significant increases in expression were observed between 2 and 4 days with decitabine and after 4 days with azacitidine. In addition, more PBD dimer drug from SGN-CD33A was incorporated into DNA in HMA treated cells. Concomitant, the treatment of AML cells with the combination of SGN-CD33A and HMA resulted in greater DNA damage and apoptosis as shown by the increased levels of the early DNA damage marker, γH2AX, and the formation of cleaved PARP, an apoptosis marker, compared to either agent alone. Studies are in progress to investigate the effects on other components of the DNA repair and cell cycle pathways.These findings demonstrate that SGN-CD33A can be successfully combined with hypomethylating agents to deliver marked antitumor activity in preclinical drug-resistant models of AML. DisclosuresSutherland:Seattle Genetics, Inc.: Employment. Yu:Seattle Genetics, Inc.: Employment. O'Day:Seattle Genetics,Inc: Employment. Alley:Seattle Genetics,Inc.: Employment. Anderson:Seattle Genetics, Inc.: Employment. Emmerton:Seattle Genetics, Inc.: Employment. Zeng:Seattle Genetics, Inc.: Employment. O'Meara:Seattle Genetics, Inc: Employment, Equity Ownership. Feldman:Seattle Genetics,Inc: Employment. Kennedy:Seattle Genetics,Inc: Employment, Equity Ownership, Honoraria, Speakers Bureau. Ryan:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc: Employment.

T-LAK cell-originated protein kinase presents a novel therapeutic target in FLT3-ITD mutated acute myeloid leukemia.

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

213 A phase I dose-finding study of BI 853520, a potent and selective inhibitor of focal adhesion kinase (FAK), in Japanese and Taiwanese patients with advanced or metastatic solid tumors

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in repressive chromatin environment that silences gene expression and has been shown to play a role in development and hematopoietic differentiation. LSD1 is overexpressed in multiple tumor types, including acute myeloid leukemia (AML). Together, these studies suggest LSD1 is an important regulator of the epigenome that modulates gene expression through modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, potent, irreversible, GSK LSD1 inhibitor (GSK2879552) in AML and small cell lung cancer (SCLC). Screening of over 150 cancer cell lines revealed that SCLC and AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlates with increased gene expression. Treatment of AML cells with GSK2879552 promotes the expression of cell surface markers associated with a differentiated immunophenotype, including CD11b and CD86. In an MV-4−11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in a subset of SCLC cell lines tested and all AML cell lines tested. Potent growth inhibition was also observed on AML blast colony forming ability of bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into secondary recipient mice that were treated with an LSD1 tool molecule inhibitor for 17 days. Control mice succumbed to AML by 45 days post transplant, while treated mice showed prolonged survival. GSK2879552 treatment of mice engrafted with SCLC cell lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

Abstract 4317: Inhibiting the mitochondrial DNA polymerase gamma (POLG) with 2′,3′-dideoxycytidine reduces oxidative phosphorylation and increases apoptosis in acute myeloid leukemia (AML) cells

Abstract Mitochondria contain their own genome and translation machinery, which are required for aerobic energy production through oxidative phosphorylation. A subset of AML patients are sensitive to inhibitors of mitochondrial translation, likely due to increased mitochondrial mass, increased mitochondrial DNA (mtDNA) and reliance on oxidative phosphorylation (Skrtic et. al., Cancer Cell, 20:674, 2011). In the current study we addressed a complementary approach of inhibiting mtDNA replication by targeting the mitochondrial DNA polymerase gamma (POLG). POLG is encoded by the nuclear genome and is the only known DNA polymerase in mammalian mitochondria. POLG indirectly controls electron transport chain (ETC) function since it replicates the mitochondrial genome that encodes 13 proteins essential for ETC activity. We explored the impact of inhibiting POLG in AML cells with the nucleoside analogue reverse transcriptase inhibitor 2′,3′-dideoxycytidine (ddC), an FDA-approved antiviral drug that demonstrates off-target inhibition of POLG. In OCI-AML2 and TEX leukemia cells, treatment with 0.2-2µM ddC reduced levels of mtDNA in a dose- and time-dependent manner, with &amp;gt;90% depletion of mtDNA after 6 days of treatment with 0.2µM ddC (p &amp;lt;0.01). mtDNA depletion resulted in decreased protein expression of the mtDNA-encoded cytochrome c oxidases (COX) 1 and 2, that form the catalytic core of ETC complex IV. In contrast, levels of COX4, a nuclear-encoded subunit of the same respiratory complex were unaltered by ddC. Likewise, 0.2 and 2µM ddC reduced mRNA transcript levels of all mtDNA-encoded proteins. ddC (2 µM, 3 days) also decreased the activity of respiratory chain complex IV by greater than 50%. Importantly, AML cells appear to have a large reserve in their mtDNA content; in both TEX and OCI-AML2 cells, mtDNA depletion down to 5% of control was required for levels of mtDNA-encoded transcripts or proteins to be significantly reduced. ddC treatments (2µM, 6-10 days) that depleted mtDNA beyond the above threshold reduced the proliferation (p&amp;lt;0.05) and increased apoptosis (p&amp;lt;0.01) of OCI-AML2 and TEX cells. In addition, 2µM ddC diminished the basal oxygen consumption rate (p&amp;lt;0.05), a measure of oxidative phosphorylation in OCI-AML2 cells and TEX cells without altering mitochondrial mass. In summary, treatment of AML cells with ddC depletes mtDNA, decreases mitochondrial bioenergetics and causes cell kill. However, AML cells have large reserves in their mtDNA content and can withstand depletion of up to 95% of their mtDNA without loss of oxidative metabolism. Citation Format: Sanduni Liyanage, Rose Hurren, Rebecca Laposa, Aaron Schimmer. Inhibiting the mitochondrial DNA polymerase gamma (POLG) with 2′,3′-dideoxycytidine reduces oxidative phosphorylation and increases apoptosis in acute myeloid leukemia (AML) cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4317. doi:10.1158/1538-7445.AM2014-4317

SMG1 acts as a novel potential tumor suppressor with epigenetic inactivation in acute myeloid leukemia.

Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently, SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2'-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.

OP449, a Novel SET Antagonist, Is Cytotoxic To Leukemia Cells and Enhances Efficacy Of Tyrosine Kinase Inhibitors In Drug-Resistant Myeloid Leukemias

BackgroundThe SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. ResultsTreatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a >2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p<0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. ConclusionsSET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures:Agarwal:Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.

The microtubule depolymerizing agent CYT997 effectively kills acute myeloid leukemia cells via activation of caspases and inhibition of PI3K/Akt/mTOR pathway proteins

The orally active microtubule-depolymerizing agent CYT997 is potently cytotoxic to a variety of tumors in vitro and in vivo. However, the effects of this agent on acute myeloid leukemia (AML) cells and its mechanisms are unknown. The present study demonstrated that CYT997 effectively inhibited the growth of AML cells in vitro. Treatment of AML cells with CYT997 resulted in G2/M phase cell cycle arrest, and induced apoptosis through the activation of extrinsic and intrinsic apoptotic pathways. Furthermore, CYT997 induced cell death in CD123+ leukemia cells and significantly reduced leukemia colony formation. CYT997 was also demonstrated to exert dual effects on the expression of PI3K/Akt and mechanistic target of rampamycin (mTOR) signaling pathway proteins. Therefore, CTY997, used alone or in combination with chemotherapy, may represent a promising approach for the treatment of AML.

Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors

AbstractMnk kinases regulate the phosphorylation and activation of the eukaryotic initiation factor 4E (eIF4E), a protein that plays key roles in the initiation of messenger RNA translation and whose activity is critical for various cellular functions. eIF4E is deregulated in acute myeloid leukemia (AML), and its aberrant activity contributes to leukemogenesis. We determined whether cercosporamide, an antifungal agent that was recently shown to act as a unique Mnk inhibitor, exhibits antileukemic properties. Treatment of AML cells with cercosporamide resulted in a dose-dependent suppression of eIF4E phosphorylation. Such suppression of Mnk kinase activity and eIF4E phosphorylation by cercosporamide resulted in dose-dependent suppressive effects on primitive leukemic progenitors (CFU-L) from AML patients and enhanced the antileukemic properties of cytarabine (Ara-C) or mammalian target of rapamycin (mTOR) complex 1 inhibition. Similarly, the combination of cercosporamide with cytarabine resulted in enhanced antileukemic responses in a xenograft mouse model in vivo. Altogether, this work demonstrates that the unique Mnk inhibitor cercosporamide suppresses phosphorylation of eIF4E and exhibits antileukemic effects, in support of future clinical-translational efforts involving combinations of Mnk inhibitors with cytarabine and/or mTOR inhibitors for the treatment of AML.

Highlights of This Issue

Acute myeloid leukemia (AML) is generally amenable to first-line chemotherapeutics that typically induce a remission. However, at least 40% of AML patients will experience a relapse, suggesting that AML cells either acquire resistance or originate from a stem cell population. Leukemia-initiating cells (LIC) are believed to be important for AML growth and are thought to be responsible for AML relapses. Therefore, strategies specifically targeting LICs are of translational interest. Weidenaar and colleagues evaluated a receptor tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK), using long-term cocultures of pediatric CD34+ AML cells. Critically, PTK/ZK inhibited outgrowth and self-renewal of LICs when cultured up to 10 weeks. Proteome profiler kinase array analysis of downstream signaling systems revealed that PTK/ZK reduced activation of phosphoinositide 3-kinase/Akt kinase signaling. Treatment of AML cells with an Akt inhibitor showed similar results, whereas antibodies against VEGFA and VEGF receptors did not. These data suggest that targeting of multiple kinase pathways with PTK/ZK could be a promising approach to improve AML outcomes.The histone methyltransferase enhancer of zeste homolog 2 (EZH2) is upregulated in many cancer types and is considered a putative therapeutic target. Garipov and colleagues report that EZH2 is upregulated in ovarian cell lines and in expression data from patients with ovarian cancer. They observed that transcriptional activation of EZH2 was dependent on 2 NF-Y binding sites in the proximal region of the EZH2 promoter. Moreover, elevated expression of NF-YA, the regulatory subunit of NF-Y, was correlated with increased EZH2 expression and EZH2 was predictive of shorter patient survival. Mechanistically, depletion of NF-YA was sufficient to decrease EZH2 levels, which phenocopy EZH2 inhibition and result in apoptosis of cancer cells. This study establishes NF-Y as a key regulator of EZH2 expression and implies that targeting NF-Y represents an alternative strategy to inhibiting EZH2 activity in cancer.The origin recognition complex (ORC), composed of multiple DNA binding proteins that associate with origins of replication, is highly regulated throughout the cell cycle to avoid re-replication and potential genomic instability. In tumorderived cells, these ORC proteins are often expressed at high levels to accommodate the stress of replication. During replication, only a fraction of origins are used, while others are thought to remain dormant, and these “dormant” replication origins have been shown to enhance recovery from replication stress. Zimmerman and colleagues show that depletion of ORC1, ORC6, or CDC6 to levels that do not affect viability, which was considered optimal for the depletion of dormant origins without affecting normal replication, hypersensitized tumor-derived cells, but not immortalized, nontransformed cells, to replication stress. Additional study revealed that altering p53 or MYC affected the sensitivity of immortalized, nontransformed cells. These findings suggest that tumor cells have a greater reliance on origin licensing capacity, revealing ORC proteins as potential targets for therapeutic intervention.Prostate cancer is a leading cause of cancer associated death in men, due largely to disseminated disease. Unfortunately, the mechanisms that promote prostate cancer metastasis are not well studied, nor does an effective treatment exist for disseminated disease. To this end, the current study focused on the expression and role of the adhesion protein, β1 integrin. Lee and colleagues determined that β1 integrin and downstream phospho-FAK were increased in both primary prostate cancer and lymph node metastases. Moreover, highly metastatic prostate cancer cells were found to have higher constitutively active β1 integrin that correlated with resistance to anoikis. Antibody blockade of β1 integrin resulted in inhibition of phenotypes, signaling, and survival processes associated with metastatic potential. Finally, systemic delivery of a neutralizing antibody to β1 integrin suppressed metastasis to lymph nodes and to bone following orthotopic and intracardiac injections, respectively. Thus, constitutively active β1 integrin is a critical effector of prostate cancer metastasis, and these preclinical findings identify a potential target for metastasis prevention.

Receptor Activator for NF-κB Ligand in Acute Myeloid Leukemia: Expression, Function, and Modulation of NK Cell Immunosurveillance

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.