Acute Myeloid Leukemia Drug Research Articles

Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03

Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

A Screening of Antineoplastic Drugs for Acute Myeloid Leukemia Reveals That Fludarabine Has Weak Immunogenic Capacity and Induces T Regulatory Cells

INTRODUCTION: Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with Daunorubicin (DNR) but not Cytarabine (Ara-C), results in efficient activation of anti-leukemia T cells. This process, named as immunogenic cell death (ICD), is characterized by some specific events. In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. However, the probability of relapse remains elevated, particularly in elderly or prognostically "high risk" patients In the last decade, Etoposide (Eto) and Fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect, and tested in many trials. Regarding the immunogenicity of these two drugs, too few studies are reported in recent literature, and even fewer regarding AML. We therefore studied the immunogenic potential of Eto and Flu as compared to DNR and Ara-C. METHODS: AML cell lines HL-60 and KG-1, and primary AML cells were treated with all four drugs. Calreticulin and heat shock proteins 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were then pulsed into dendritic cells and used forin vitroimmunological tests, in particular for T-cell proliferation and T regulatory cells (Tregs) induction. RESULTS: Collectively, our data indicate that, among the drugs that have been proposed to increase the efficacy of the conventional chemotherapy backbone including DNR and Ara-C, Eto has a similar and comparable capacity to DNR in inducing both early and late ICD events. On the contrary, Flu has a low if any effect, proving similar to Ara-C. Moreover, Eto treatment was the most powerful among the tested drugs in stimulating T-cell proliferation, thus suggesting a significant capacity to activate the immune response. On the contrary, Flu had weak immunogenic potential and can be considered a non-immunogenic chemotherapy drug. Interestingly, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs. CONCLUSIONS: Taken together, the present investigation expands the knowledge on the immunogenic and tolerogenic potential of the chemotherapy drugs commonly used in the therapy of AML. Among these, important differences have been observed, indicating that, particularly in an era when immunotherapy is being included in the clinical stage of AML treatment, the immunological perspective of chemotherapy should be taken into consideration in therapy decision-making. This research was funded by AIRC (Associazione Italiana per la Ricerca sul Cancro) 2017 IG20654. Bologna AIL (Associazione Italiana contro le Leucemie)/ Bologna Section. FATRO/Foundation Corrado and Bruno Maria Zaini-Bologna. Disclosures Cavo: Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Janssen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau;GlaxoSmithKline:Honoraria, Speakers Bureau;Karyopharm:Honoraria;Novartis:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Celgene:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau;AbbVie:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;BMS:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Targeting E-Selectin with GMI-1271 Overcomes Microenvironment-Mediated Resistance to Venetoclax/HMA Therapy

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (Ven/HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Despite improved remission rates, the duration of response is still inadequate. Adhesion to the bone marrow (BM) niche is critical for AML initiation, progression and leukemic stem cell (LSC) survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) is responsible for the tethering and rolling of leukocytes on perivascular endothelial BM niche cells (EC). In leukemia, E-sel has crucial roles in BM homing and engraftment (Krause et al, 2006). Through patient-derived AML xenograft (PDX) models and single cell proteomics, we have elucidated the roles of E-sel in AML survival and drug resistance. A PDX model derived from a patient who had developed resistance to Ven/HMA were treated with the E-sel antagonist, GMI-1271 (uproleselan; GlycoMimetic, Inc). We found that targeting E-sel mobilized human AML cells and sensitized them to Ven/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with Ven/HMA in comparison to Ven/HMA alone (p < 0.05). The synergistic effects of the combinatorial treatment on AML-PDX mouse survival were determined by Kaplan-Meier analysis. The combination of GMI-1271 and Ven/HMA significantly prolonged the survival of mice compared to vehicle control (p = 0.015) as well as the Ven/HMA (p = 0.0009) and GMI-1271 groups (p = 0.03). The median survival of the vehicle control, GMI-1271, Ven/HMA, and combination-treated groups of mice was 86, 91, 81.5, and 106.5 days, respectively. Histological analysis of BM, spleen, lung and liver demonstrated differences in leukemia cell infiltration, confirming enhanced anti-leukemia efficacy of the combination treatment. To delineate the mechanism of E-sel at the onset of drug mediated changes in AML signaling signatures, we employed another PDX model (Flt3-ITD and WT1 mutations, sorafenib-resistant). PDX mice with advanced AML (more than 20% human AML cells circulation in peripheral blood) were administered Ven/HMA, GMI-1271, or combination for 2 days. Single cell proteomics analysis by CyTOF determined that combinatorial treatment diminished levels of Ki67, IDU, and pRb compared to vehicle control or Ven/HMA alone, resulting in decreased proliferation of AML blasts. Activation of eNOS to produce nitric oxide (NO) through PI3K/AKT kinase, maintains clonogenic cell growth in malignant cells. A recent publication has demonstrated that introduction of NOS blockers in combination with chemotherapy led to slower leukemia progression and longer remissions in contrast to chemotherapy alone (Passaro et al, 2017). Interestingly, we observed reduced activation of PI3K and AKT in AML blasts as well as in BM CD31+EC cells in the GMI-1271 treated PDX model. eNOS phosphorylation was subsequently decreased in EC, suggesting that inhibition of E-sel may protect BM vasculature by blocking the production of NO. In addition, targeting E-sel showed signaling alterations in AML MSC. Administration of E-sel antagonist increased mTOR expression in MSC from AML-PDX. Combination treatment induced higher Ki67 positivity, and hyperactivation of pRb and p-S6 in MSC in vivo. Our group and others have recently reported that Ven-resistant AML cells exhibit an increased dependence on alternate anti-apoptotic proteins, Mcl-1 and Bcl-xl (Konopleva et al., 2016). We found that concomitant treatment in vivo with GMI-1271 and Ven/HMA further decreased the expression of Bcl-xl and Mcl-1 in AML blasts compared to Ven/HMA alone, suggesting a critical role for E-sel antagonists in overcoming drug resistance. E-selectin binding potential and focal adhesion kinase activity in AML blasts were decreased upon acute administration of pharmacological E-sel inhibitor. Other oncogenic signaling pathways including MAPK, p-S6, and STAT3, were all inhibited by the addition of GMI-1271 to Ven/HMA. Collectively, our results provide first evidence that an E-sel targeting strategy with GMI-1271 can overcome microenvironmental resistance to Ven/HMA-based therapy in AML by cancer cell autonomous and non-cell autonomous mechanisms in the BM vascular niche. Figure Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Priothera raises funds for leukemia therapy

Priothera has raised about $35 million in series A financing to develop drugs for acute myeloid leukemia (AML). The Dublin-based company will use the funds to test mocravimod in a clinical trial of...

A Screening of Antineoplastic Drugs for Acute Myeloid Leukemia Reveals Contrasting Immunogenic Effects of Etoposide and Fludarabine.

Background: Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with daunorubicin (DNR) but not cytarabine (Ara-C) results in immunogenic cell death (ICD). In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. In the last decade, etoposide (Eto) and fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect and have been tested in many trials. Very little data are available about the ability of these drugs to induce ICD. Methods: AML cells were treated with all four drugs. Calreticulin and heat shock protein 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were pulsed into dendritic cells (DCs) and used for in vitro immunological tests. Results: Flu and Ara-C had no capacity to induce ICD-related events. Interestingly, Eto was comparable to DNR in inducing all ICD events, resulting in DC maturation. Moreover, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs. Conclusions: Our results indicate a novel and until now poorly investigated feature of antineoplastic drugs commonly used for AML treatment, based on their different immunogenic potential.

CD33-Targeted Therapies: Beating the Disease or Beaten to Death?

CD33 is a transmembrane protein that is found on cells of myeloid lineage. It is also intensely expressed on acute myeloid leukemia (AML) progenitor cells but not on normal stem cells. It internalizes on binding and dimerization, making it a specific and ideal target for AML therapeutics and drug delivery. Several targeted therapies have been tested and many are still currently in development. Gemtuzumab ozogamicin was the first and only CD33-directed antibody-drug conjugate to be US Food and Drug Administration approved for AML. Other targeted agents have not achieved such success. Promising new strategies include cellular therapy mechanisms and linker molecules. This is an exciting target that requires a considerable amount of precision to yield clinical benefit.

Synergistic Anti Leukemia Effect of a Novel Hsp90 and a Pan Cyclin Dependent Kinase Inhibitors.

Acute myeloid leukemia (AML) is among the top four malignancies in Saudi nationals, and it is the top leukemia subtype worldwide. Resistance to available AML drugs requires the identification of new targets and agents. Hsp90 is one of the emerging important targets in AML, which has a central role in the regulation of apoptosis and cell proliferation through client proteins including the growth factor receptors and cyclin dependent kinases. The objective of the first part of this study is to investigate the putative Hsp90 inhibition activity of three novel previously synthesized quinazolines, which showed HL60 cytotoxicity and VEGFR2 and EGFR kinases inhibition activities. Using surface plasmon resonance, compound 1 (HAA2020) showed better Hsp90 inhibition compared to 17-AAG, and a docking study revealed that it fits nicely into the ATPase site. The objective of the second part is to maximize the anti-leukemic activity of HAA2020, which was combined with each of the eleven standard inhibitors. The best resulting synergistic effect in HL60 cells was with the pan cyclin-dependent kinases (CDK) inhibitor dinaciclib, using an MTT assay. Furthermore, the inhibiting effect of the Hsp90α gene by the combination of HAA2020 and dinaciclib was associated with increased caspase-7 and TNF-α, leading to apoptosis in HL60 cells. In addition, the combination upregulated p27 simultaneously with the inhibition of cyclinD3 and CDK2, leading to abolished HL60 proliferation and survival. The actions of HAA2020 propagated the apoptotic and cell cycle control properties of dinaciclib, showing the importance of co-targeting Hsp90 and CDK, which could lead to the better management of leukemia.

LncRNA MEG3 contributes to drug resistance in acute myeloid leukemia by positively regulating ALG9 through sponging miR-155.

The development of drug resistance is the main obstacle for successful treatment in acute myeloid leukemia (AML). Noncoding RNAs have been implicated in biological function in AML drug resistance. Aberrant protein glycosylation is associated with AML progression. The aim of the study was to explore the potential regulatory mechanism of lncRNA MEG3/miR-155/ALG9 axis in drug resistance of AML. QRT-PCR and Western blot were used for comparison analyses of ALG9, MEG3, and miR-155 levels. CCK-8 and colony formation assays were determined for drug sensitivity and proliferative capability of AML cells. Luciferase reporter assay was used to confirm the targets of miR-155. The mannosyltransferase ALG9 and MEG3 was downregulated in peripheral blood mononuclear cells (PBMCs) of M5/multidrug resistance (MDR) AML patients and adriamycin (ADR)-resistant AML cell lines, which determined a positive correlation in AML patients. Low expression of ALG9 and MEG3 predicted poor prognosis of AML patients. The altered level of ALG9 was found corresponding to the drug-resistant phenotype and sphere formation of AML cells. MiR-155 was overexpressed in M5/MDR patients and ADR-resistant AML cells, as well as inversely correlated to ALG9 expression. MEG3 was a direct target of miR-155 and could sponge miR-155 in AML cells. MEG3 interacted with miR-155 to regulate ALG9 expression, which reversed the effects of ALG9 regulation on proliferation and drug resistance in AML cells. MEG3 sponged miR-155 by competing endogenous RNA (ceRNA) mechanism, which further modulated ALG9 expression and AML procession, providing a novel therapeutic target for AML chemoresistance.

FLT3 inhibition upregulates HDAC8 via FOXO to inactivate p53 and promote maintenance of FLT3-ITD+ acute myeloid leukemia

AbstractInternal tandem duplication (ITD) mutations within the FMS-like receptor tyrosine kinase-3 (FLT3) can be found in up to 25% to 30% of acute myeloid leukemia (AML) patients and confer a poor prognosis. Although FLT3 tyrosine kinase inhibitors (TKIs) have shown clinical responses, they cannot eliminate primitive FLT3-ITD+ AML cells, which are potential sources of relapse. Therefore, elucidating the mechanisms underlying FLT3-ITD+ AML maintenance and drug resistance is essential to develop novel effective treatment strategies. Here, we demonstrate that FLT3 inhibition induces histone deacetylase 8 (HDAC8) upregulation through FOXO1- and FOXO3-mediated transactivation in FLT3-ITD+ AML cells. Upregulated HDAC8 deacetylates and inactivates p53, leading to leukemia maintenance and drug resistance upon TKI treatment. Genetic or pharmacological inhibition of HDAC8 reactivates p53, abrogates leukemia maintenance, and significantly enhances TKI-mediated elimination of FLT3-ITD+ AML cells. Importantly, in FLT3-ITD+ AML patient–derived xenograft models, the combination of FLT3 TKI (AC220) and an HDAC8 inhibitor (22d) significantly inhibits leukemia progression and effectively reduces primitive FLT3-ITD+ AML cells. Moreover, we extend these findings to an AML subtype harboring another tyrosine kinase–activating mutation. In conclusion, our study demonstrates that HDAC8 upregulation is an important mechanism to resist TKIs and promote leukemia maintenance and suggests that combining HDAC8 inhibition with TKI treatment could be a promising strategy to treat FLT3-ITD+ AML and other tyrosine kinase mutation–harboring leukemias.

PERK/NRF2 and autophagy form a resistance mechanism against G9a inhibition in leukemia stem cells

BackgroundThe histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML). However, the effect of G9a inhibition on leukemia stem cells (LSCs), which are responsible for AML drug resistance and recurrence, is unclear. In this study, we investigated the underlying mechanisms of the LSC resistance to G9a inhibition.MethodsWe evaluated the effects of G9a inhibition on the unfolded protein response and autophagy in AML and LSC-like cell lines and in primary CD34+CD38− leukemic blasts from patients with AML and investigated the underlying mechanisms. The effects of treatment on cells were evaluated by flow cytometry, western blotting, confocal microscopy, reactive oxygen species (ROS) production assay.ResultsThe G9a inhibitor BIX-01294 effectively induced apoptosis in AML cell lines; however, the effect was limited in KG1 LSC-like cells. BIX-01294 treatment or siRNA-mediated G9a knockdown led to the activation of the PERK/NRF2 pathway and HO-1 upregulation in KG1 cells. Phosphorylation of p38 and intracellular generation of reactive oxygen species (ROS) were suppressed. Pharmacological or siRNA-mediated inhibition of the PERK/NRF2 pathway synergistically enhanced BIX-01294-induced apoptosis, with suppressed HO-1 expression, increased p38 phosphorylation, and elevated ROS generation, indicating that activated PERK/NRF2 signaling suppressed ROS-induced apoptosis in KG1 cells. By contrast, cotreatment of normal hematopoietic stem cells with BIX-01294 and a PERK inhibitor had no significant proapoptotic effect. Additionally, G9a inhibition induced autophagy flux in KG1 cells, while autophagy inhibitors significantly increased the BIX-01294-induced apoptosis. This prosurvival autophagy was not abrogated by PERK/NRF2 inhibition.ConclusionsPERK/NRF2 signaling plays a key role in protecting LSCs against ROS-induced apoptosis, thus conferring resistance to G9a inhibitors. Treatment with PERK/NRF2 or autophagy inhibitors could overcome resistance to G9a inhibition and eliminate LSCs, suggesting the potential clinical utility of these unique targeted therapies against AML.

Advances in the Treatment of Acute Myeloid Leukemia: New Drugs and New Challenges.

The therapeutic armamentarium of acute myeloid leukemia (AML) has rapidly expanded in the past few years, driven largely by translational research into its genomic landscape and an improved understanding of mechanisms of resistance to conventional therapies. However, primary and secondary drug resistance remains a substantial problem for most patients. Research into the mechanisms of resistance to these new agents is informing the development of the next class of AML drugs and the design of combination regimens aimed at optimally exploiting therapeutic vulnerabilities, with the ultimate goal of eradicating all subclones of the disease and increasing cure rates in AML. SIGNIFICANCE: AML is a heterogeneous disease, characterized by a broad spectrum of molecular alterations that influence clinical outcomes and also provide potential targets for drug development. This review discusses the current and emerging therapeutic landscape of AML, highlighting novel classes of drugs and how our expanding knowledge of mechanisms of resistance are informing future therapies and providing new opportunities for effective combination strategies.

Driver mutations in acute myeloid leukemia.

The mutational landscape of acute myeloid leukemia (AML) has revised diagnostic, prognostic, and therapeutic schemata over the past decade. Recurrently mutated AML genes have functional consequences beyond typical oncogene-driven growth and loss of tumor suppresser function. Large-scale genomic sequencing efforts have mapped the complexity of AML and trials of mutation-based targeted therapy has led to several FDA-approved drugs for mutant-specific AML. However, many recurrent mutations have been identified across a spectrum from clonal hematopoiesis to myelodysplasia to overt AML, such as effectors of DNA methylation, chromatin modifiers, and spliceosomal machinery. The functional effects of these mutations are the basis for substantial discovery. Understanding the molecular and pathophysiologic functions of key genes that exert leukemogenic potential is essential towards translating these findings into better treatment for AML.

Repurposing Drugs for Acute Myeloid Leukemia: A Worthy Cause or a Futile Pursuit?

Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid progenitor cells that affects patients of all ages. Despite decades of research and improvement in overall outcomes, standard therapy remains ineffective for certain subtypes of AML. Current treatment is intensive and leads to a number of secondary effects with varying results by patient population. Due to the high cost of discovery and an unmet need for new targeted therapies that are well tolerated, alternative drug development strategies have become increasingly attractive. Repurposing existing drugs is one approach to identify new therapies with fewer financial and regulatory hurdles. In this review, we provide an overview of previously U.S. Food and Drug Administration (FDA) approved non-chemotherapy drugs under investigation for the treatment of AML.

DNA-PK Inhibitor, M3814, as a New Combination Partner of Mylotarg in the Treatment of Acute Myeloid Leukemia.

Despite significant advances in the treatment of acute myeloid leukemia (AML) the long-term prognosis remains relatively poor and there is an urgent need for improved therapies with increased potency and tumor selectivity. Mylotarg is the first AML-targeting drug from a new generation of antibody drug conjugate (ADC) therapies aiming at the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic payload, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSB by the non-homologous end joining pathway driven by DNA-dependent protein kinase (DNA-PK) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg and a potential new combination treatment option for AML patients.

"Amyloid-beta accumulation cycle" as a prevention and/or therapy target for Alzheimer's disease.

The cell cycle and its regulators are validated targets for cancer drugs. Reagents that target cells in a specific cell cycle phase (e.g., antimitotics or DNA synthesis inhibitors/replication stress inducers) have demonstrated success as broad‐spectrum anticancer drugs. Cyclin‐dependent kinases (CDKs) are drivers of cell cycle transitions. A CDK inhibitor, flavopiridol/alvocidib, is an FDA‐approved drug for acute myeloid leukemia. Alzheimer's disease (AD) is another serious issue in contemporary medicine. The cause of AD remains elusive, although a critical role of latent amyloid‐beta accumulation has emerged. Existing AD drug research and development targets include amyloid, amyloid metabolism/catabolism, tau, inflammation, cholesterol, the cholinergic system, and other neurotransmitters. However, none have been validated as therapeutically effective targets. Recent reports from AD‐omics and preclinical animal models provided data supporting the long‐standing notion that cell cycle progression and/or mitosis may be a valid target for AD prevention and/or therapy. This review will summarize the recent developments in AD research: (a) Mitotic re‐entry, leading to the “amyloid‐beta accumulation cycle,” may be a prerequisite for amyloid‐beta accumulation and AD pathology development; (b) AD‐associated pathogens can cause cell cycle errors; (c) thirteen among 37 human AD genetic risk genes may be functionally involved in the cell cycle and/or mitosis; and (d) preclinical AD mouse models treated with CDK inhibitor showed improvements in cognitive/behavioral symptoms. If the “amyloid‐beta accumulation cycle is an AD drug target” concept is proven, repurposing of cancer drugs may emerge as a new, fast‐track approach for AD management in the clinic setting.

Chidamide Enhances the Cytotoxicity of Cytarabine and Sorafenib in Acute Myeloid Leukemia Cells by Modulating H3K9me3 and Autophagy Levels.

Previous studies showed that chidamide enhances the cytotoxicity of drugs in acute myeloid leukemia (AML) cells. Therefore, we examined whether chidamide enhanced the cytotoxicity of drugs in AML cells by affecting H3K9me3 and autophagy levels. AML cells (THP-1 and MV4-11 cells) were treated with chidamide, cytarabine (Ara-c), or sorafenib alone or in combination. Cell proliferation and survival rates were analyzed by MTT, flow cytometry, and Western blotting assays. The results showed that a low dose of chidamide enhanced the cytotoxicity of Ara-c or sorafenib in AML cells, decreasing proliferation and increasing apoptosis. H3K9me3 levels as assessed by Western blotting were upregulated by chidamide treatment. Chromatin immunoprecipitation sequencing, which was used to investigate potential signaling pathways, indicated that the autophagy pathway might play a role in the effects of chidamide. The level of autophagy induced in AML cells upon treatment with Ara-c or sorafenib was inhibited by chidamide, and autophagy markers (LC3, P62) were tested by Western blotting. SIRT1 messenger RNA (mRNA) and protein levels were lower in AML cells treated with Ara-c or sorafenib in combination with chidamide than those in cells treated with these drugs alone. Additionally, the Integrative Genomics Viewer results indicate that the H3K9me3 changes were related to SIRT1-binding sites. Together, these results show that chidamide enhances the cytotoxicity of two chemotherapy drugs in AML cells by increasing the H3K9me3 level and inhibiting autophagy via decreasing the expression of SIRT1. Chidamide may be a potential treatment strategy for AML in the future, especially for refractory AML patients.

An Evaluation of Major Comorbidities and Treatment Patterns of Newly Diagnosed Acute Myeloid Leukemia Patients: A Retrospective Analysis of Electronic Medical Records from US

Background: There is limited data available on patient characteristics and treatment patterns for patients diagnosed with acute myeloid leukemia (AML) particularly from electronic medical record (EMR) database in US. Objective: Our goal was to examine comorbidities and treatment patterns among newly diagnosed AML patients in US. Methods: We conducted a retrospective cohort study using fully adjudicated data provided by IQVIA from the EMR database including patient data from January 2012-July 2018 across the US. ICD-9/10-CM codes were used to identify newly diagnosed AML (excluding APL) patients. Initial AML diagnosis codes which indicated relapse or remission were excluded from the newly diagnosed population. Demographics and comorbidities at baseline as well as AML treatments received after initial diagnosis were examined. Results: A total of 3387 patients who had an AML diagnosis were retrieved. 2879 were newly diagnosed with mean age of 65.9 and median age at 70 (ranged from 17 to 85) and 54.8 % were male. Prior to the first AML diagnosis record (i.e. index date), 18.7% had other prior malignant solid tumors and 13.5% had antecedent Myelodysplastic Syndrome (MDS), while 71.2% were AML de novo. Cardiovascular diseases (18.7%) and diabetes (4.6%) were the most common comorbidities occurred before or after initial AML diagnosis. After index date, 38.2% of the newly diagnosed patients received FDA approved drugs for AML and 18% received supportive care only. 2.3 % received hematopoietic cell transplant. Among the FDA approved drugs for AML, hypomethylating agents (37.4% Decitabine and 35.3% Azacitidine), cytarabine based treatments (21.8%) were the most commonly used first line treatment. 25% switched to other AML drugs later in the treatment. Filgrastim (49.6%) and Azoles (37.4%) were the most frequently used supportive care. Conclusions: Among newly diagnosed AML patients, most had AML de novo. Cardiovascular disease and diabetes are the most common comorbidities and monotherapy was predominantly used as first line therapy. Low percentage of hematopoietic cell transplant observed in this population needs further investigation. Disclosures Tu: Daiichi Sankyo, Inc.: Employment. Islam:Daiichi Sankyo, Inc.: Employment. Fliss:Daiichi Sankyo, Inc.: Employment. Salas:Daiichi Sankyo, Inc.: Employment.

A Population-Based Study of T/Myeloid Mixed Phenotype Acute Leukemia

Introduction: Mixed phenotype acute leukemia (MPAL) is exceeding rare, making up less than 1% of all acute leukemias1, 2. It harbors features of both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Most cases have features of B-ALL; those with features of T-ALL are particularly uncommon2. We treated a 74-year-old retired male with T/myeloid MPAL. His disease progressed through 7 lines of therapy over 18 months, resulting in significant cytopenias and toxicity. Given the refractory nature of his disease to numerous ALL and AML-inspired regimens, we wanted to better understand trends in survival for this rare disease. Methods: The Surveillance, Epidemiology, and End-Result (SEER)-18 database was utilized, which encompasses 28% of the U.S. population. Patients of all ages diagnosed with T/myeloid MPAL between 2010-2016 were included. Patients with T-ALL and AML were used as comparators. The primary endpoint was disease-specific survival. Variables assessed included age, race/ethnicity, insurance type, geographical location, rural vs. urban location and socioeconomic status using county level data. Categorical variables were compared with the chi-square and fisher's exact test and survival was characterized with the cox proportional hazard model. This study was exempt from the IRB. Results: Ninety-five cases of T/myeloid MPAL were reported from 2010-2016 through the SEER-18 database. T/myeloid MPAL demonstrated a broad age distribution (median age: 40, IQR: 17-61), in comparison to T-ALL which mostly affected adolescence/young adults (median age: 17.5, IQR: 9-35) and AML which mostly affected older adults (median age: 67, IQR: 53-78) (figure 1). The 5-year survival was 50.4%, which was higher than AML (23.3%) and lower than T-ALL (70.6%) (figure 2). Survival did not change from 2010 to 2016. After adjusting for age, race/ethnicity, insurance type, geographical location, urban vs. rural location, and county level surrogates for poverty, the only two variables that significantly impacted survival were increasing age (HR 3.41, 95%CI: 2.13-5.46, p<.0001) and Medicaid insurance (HR 3.2, 95%CI: 1.27-8.09, p=.014) (figure 3-4). Estimated 5-year survival ranged from over 70% for children, adolescence and young adults to only 16% for older adults. Discussion: T/myeloid MPAL affects people of all ages. Given its rarity, there are no actively enrolling clinical trials or randomized controlled trials from which to guide management. Treatments are largely extrapolated from T-ALL and AML2. Despite newer FDA approved drugs for T-ALL and AML and improvements in allotransplant, survival has not improved and remains dismal for elderly patients. Older patients may have more aggressive disease biology and are often not candidates for intensive chemotherapy or allotransplant due to comorbidities and diminished reserve. Patient age very likely contributes to the difference in survival between T/myeloid MPAL and T-ALL or AML however Medicaid insurance also adversely affected survival in T/myeloid MPAL. This etiology is unclear, however could relate to decreased access to care. Conclusion: T/myeloid MPAL is an exceedingly rare disease with dismal outcomes in the elderly. A better understanding of the disease biology in elderly patients, insurance related barriers to care, and more personalized treatments are needed to improve patient outcomes.

Role of HDAC8 Upregulation in Resistance of FLT3-ITD AML Stem Cells to FLT3 Inhibitors

The FLT3-ITD mutation occurs in 25-30% of acute myeloid leukemia (AML) incidence, and is associated with higher relapse rate and inferior survival. FLT3 inhibitors have shown potent antileukemic effect, but the responses are not sustained in most FLT3-ITD AML patients. Histone deacetylases (HDACs) are crucial for cancer stem cell maintenance through epigenetically regulating gene expression, modulating the function of non-histone proteins via post-translational modification, and maintaining genome stability. Here we investigated the potential role of HDACs in FLT3 inhibitors resistance and FLT3-ITD AML stem cells maintenance. We firstly evaluated mRNA expression alterations of 11 HDACs in FLT3-ITD+ MV4-11 and MOLM-13 AML cells upon FLT3 inhibitor AC220 (Quizartinib) treatment, and found that HDAC8 was the most significantly upregulated gene. Inhibition of FLT3 signaling via siRNA-mediated knockdown of FLT3 also resulted in increased expression of HDAC8. Treatment of primary FLT3-ITD+ CD34+ AML cells with AC220 confirmed HDAC8 upregulation. Targeting HDAC8 using specific HDAC8 inhibitor 22d or knockdown of HDAC8 readily resulted in significant apoptosis of cell lines and primary AML samples, and inhibited colony growth and replating activity in primary FLT3-ITD+ CD34+ AML cells. Moreover, these antileukemic effects were even more pronounced upon 22d and AC220 combination. To explore the mechanism of HDAC8 mediated FLT3 inhibitor resistance, we investigated transcriptomic change of MV4-11 cells upon 22d treatment by RNA-seq. KEGG pathway analysis revealed that the p53 signaling pathway was significantly upregulated. Upstream regulator prediction by Ingenuity pathway analysis also showed p53 as the key activated upstream transcription factor. Western blot analysis on acetylated p53 (Lys382) demonstrated that targeting FLT3 signaling had little effect on the p53 acetylation, while suppression of HDAC8 markedly increased p53 acetylation, and this effect was even more significant when both FLT3 and HDAC8 were inhibited simultaneously. Knockdown of p53 could partially rescued FLT3-ITD AML cells from 22d and/or AC220 induced apoptosis, and offset the inhibitory effect on growth and colony formation. We next studied the mechanism of HDAC8 upregulation upon FLT3 signally inhibition. We found FOXO1 and FOXO3 (FOXOs) binding motifs in HDAC8 promoter. FLT3 signaling inhibition promoted the binding of FOXOs to HDAC8 promoter. Knockdown both FOXOs abrogated HDAC8 upregulation upon AC220 treatment, and caused growth inhibition and apoptosis of FLT3-ITD AML cells. Finally, to translate our experimental results into clinical usage, we tested the in vivo effect of 22d, AC220, and the combination of both on patient-derived xenograft (PDX) model engrafted with four primary FLT3-ITD AML samples. 22d or AC220 treatment reduced the percentage and total number of human CD45+ leukemic cells in murine bone marrow, and prolonged the survival of leukemic mice. The combination of 22d and AC220 significantly reduced AML cell engraftment compared with either 22d or AC220 alone, and further elongated the survival period. Human CD34+ cells in BM were markedly reduced in mice treated with 22d or AC220 alone, with further reduction seen in combination. Secondary transplantation of BM cells from mice receiving combination treatment resulted in significantly reduced engraftment compared with AC220 treatment alone, indicating reduced LSC capacity of residual cells. These results show that combination of 22d and AC220 enhances targeting of primitive AML stem cells in vivo. In conclusion, upon FLT3 inhibitor treatment, FLT3-ITD AML cells may upregulate the expression of HDAC8 through FOXOs activation, which in turn inhibit the function of tumor suppressor gene p53, and promote the maintenance of FLT3-ITD AML stem cells and drug resistance. Targeting FLT3 and HDAC8 simultaneously may be promising approach to eliminating FLT3-ITD AML stem cells, and to cure this subset of leukemia. Disclosures No relevant conflicts of interest to declare.

LAM-003, a Novel Oral Heat Shock Protein 90 Inhibitor for Treatment of Acute Myeloid Leukemia, Including Wild-Type and FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Disease

Acute myeloid leukemia (AML) remains a disease with high unmet medical need. While most patients respond to initial therapy, few are cured, relapse rates are high, and most patients eventually develop life-threatening complications. FLT3-mutant disease is a particularly aggressive subtype. Recent approval of drugs targeting FLT3-mutant disease have improved short-term outcomes but not all patients respond, and duration of response is limited by secondary mutations that impede FLT3 inhibitor (FLT3i) binding (intrinsic factors) and by secreted stromal factors that activate alternative pro-survival pathways (extrinsic factors). Heat-shock protein 90 (HSP90) is a chaperone protein involved in many cellular processes and inhibition of HSP90 can have pleiotropic effects in targeting cancer cells such as degradation of oncoproteins that drive survival and proteins that mediate protective signaling. Here, we describe the nonclinical activity of LAM-003, an orally bioavailable HSP90 inhibitor (HSP90i) under clinical development for AML. To assess the anti-leukemic activity of LAM-003, we tested a panel of AML cell lines and primary AML samples. LAM-003 inhibited proliferation of both FLT3-mutant and wild-type cell lines, with preferential activity against cells harboring FLT3-ITD (geometric mean FLT3-ITD EC50 = 670 nM [n=8] vs FLT3 WT EC50 = 1400 nM [n=16]). Additionally, we observed that LAM-003 was potent in a subset of the FLT3 WT cells. To explore whether LAM-003 was effective against tumors driven by oncoproteins that are client proteins of HSP90, we focused on AML cells harboring FLT3-ITD. We confirmed that LAM-003 reduced cell surface FLT3-ITD expression and downstream signaling in MV-4-11 and MOLM-13 cells, consistent with HSP90i-mediated degradation of FLT3-ITD. In BA/F3 cells expressing FLT3-ITD with various secondary resistance mutations, we observed that LAM-003 elicited a dose-dependent reduction of FLT3 mutant cell surface expression. Moreover, BA/F3 cells expressing FLT3-ITD and the F691L mutation exhibited the expected resistance to crenolanib, yet LAM-003 retained anti-proliferative activity. Additionally, MOLM-13 cells harboring a FLT3 D835Y mutation demonstrated expected resistance to the FLT3i sorafenib and tandutinib yet remained sensitive to LAM-003. Finally, primary AML blasts harboring a D835 mutation displayed sensitivity to LAM-003 when tested ex vivo. To evaluate the potential of LAM-003 to overcome bone-marrow-stroma-derived resistance, FLT3-ITD AML cell lines (MV-4-11, MOLM-13, MOLM-14) were assayed in unconditioned or stromal-cell-conditioned medium. Conditioned medium dramatically reduced the potency of FLT3i but LAM-003 demonstrated equal potency under both conditions. We also showed that stromal cell co-culture induced FLT3i resistance in MOLM-13 cells whereas LAM-003 retained potent activity. Recognizing that the inherent genetic heterogeneity of AML blasts limits the curative potential of a single drug, we performed in vitro studies to identify drugs that synergize with LAM-003 in 3 FLT3-ITD AML cell lines. Synergy was demonstrated with FLT3i, daunorubicin, azacitidine or cytarabine, with the most robust synergy being observed with venetoclax. Extending the evaluation to AML cells wild type for FLT3 and cell lines from other hematologic indications (multiple myeloma, diffuse large B-cell lymphoma and mantle cell lymphoma), we found that the synergy was not limited to cells harboring FLT3-ITD, but rather correlated with BCL-2 abundance, suggesting a fundamental mechanism of action that depends on BCL-2 family-mediated survival. Mechanistic studies demonstrated that the combination of LAM-003 and venetoclax inhibited AKT-mediated regulation of GSK3B, resulting in MCL-1 degradation. In vivo studies using a MOLM-13 systemic model of FLT3-ITD AML demonstrated that LAM-003 monotherapy significantly improved animal survival and that the combination of LAM-003 and venetoclax significantly prolonged animal survival compared with each single agent. These nonclinical studies demonstrate that LAM-003 exhibits antileukemic activity, overcomes mechanisms of FLT3i resistance and potently synergizes with existing AML drugs. As such, our data provide strong rationale for evaluation of LAM-003 in an ongoing clinical trial in patients with AML (NCT03426605). Disclosures Beeharry: AI Therapeutics: Employment, Equity Ownership. Landrette:AI Therapeutics: Employment. Grotzke:AI Therapeutics: Employment. Gayle:AI Therapeutics: Equity Ownership. Young:AI Therapeutics: Employment, Equity Ownership. Miller:Incuron, Inc.: Consultancy; Cleveland Biolabs, Inc: Employment, Equity Ownership; Calistoga Pharmaceuticals, Inc.: Equity Ownership; AI Therapeutics: Consultancy, Equity Ownership; VelosBio Inc.: Employment, Equity Ownership; Acerta Pharma, Inc.: Equity Ownership. Xu:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rothberg:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lichenstein:AI Therapeutics: Employment, Equity Ownership.