Efficacy In Acute Myeloid Leukemia Research Articles

GPR132 Has Therapeutic Efficacy in Acute Myeloid Leukemia in Vivo by Promoting Cellular Differentiation in an mTOR Dependent Way: Identification of 8-Gingerol as a Novel Agonist

GPR132 Has Therapeutic Efficacy in Acute Myeloid Leukemia in Vivo by Promoting Cellular Differentiation in an mTOR Dependent Way: Identification of 8-Gingerol as a Novel Agonist

Targeting CD38 in acute myeloid leukemia interferes with leukemia trafficking and induces phagocytosis

Targeting the interaction between leukemic cells and the microenvironment is an appealing approach to enhance the therapeutic efficacy in acute myeloid leukemia (AML). AML infiltration induces a significant release of inflammatory cytokines in the human bone marrow niche which accelerates leukemogenesis. As the transmembrane glycoprotein CD38 has been shown to regulate cytokine release, we assessed the anti-leukemic potential of CD38 inhibition in AML. CD38 expression in AML cells proved to depend on microenvironmental cues and could be significantly enforced through addition of tretinoin. In fact, the anti-CD38 antibody daratumumab showed significant cytostatic efficacy in a 3D in vitro triple-culture model of AML, but with modest cell-autonomous cytotoxic activity and independent of CD38 expression level. In line with a predominantly microenvironment-mediated activity of daratumumab in AML, CD38 inhibition significantly induced antibody-dependent phagocytosis and showed interference with AML cell trafficking in vivo in a xenograft transplantation model, but overall lacked robust anti-leukemic effects.

A Prospective Clinical Study of the Efficacy and Adverse Reactions of Azacitidine Combined with IA Regimen As the Induction Treatment of Newly Diagnosed AML

Objective:This single-arm prospective research (ChiCTR2100044731) aimed to evaluate the efficacy and safety of azacitidine combined with IA regimen in the induction treatment of newly diagnosed acute myeloid leukemia (AML), with a view to further improving the efficacy of acute myeloid leukemia with poor prognosis.Methods:Newly diagnosed AML (non-M3) patients who received azacitidine combined with IA regimen induction chemotherapy in the Department of Hematology of the First Affiliated Hospital of Sun Yat-sen University from November 2019 to February 2021 were enrolled, and the efficacy and side effect were analyzed.Results: 1.A total of 33 patients were enrolled. The median age of the enrolled patients was 43.36 years (17-63), including 16 males (48.5%) and 17 females (51.5%). According to NCCN risk stratification, there were 3 patients (9.1%) in the favor group (9.1%) ,13 cases (39.4%) in the intermediate group and 17 cases (51.5%) in the poor group.2.The CR rate of one cycle of azacitidine combined with IA regimen was 66.7%, with a PR rate of 12.1% and a NR rate of 21.2%. After propensity score matching with the newly diagnosed AML patients who received IA regimen as induction chemotherapy in our center, a paired study was carried out. The results showed that there was no significant difference between the 2 groups in the treatment CR rate (66.7% for azacitidine combined with IA vs 54.5% for IA, P=0.592, Fig1). Subgroup analysis (table 1) showed combination of azacitidine with IA significantly improved the CR rate of patients with a ratio of blasts in the bone marrow greater than 67% (83.3% vs 30.8%, P=0.014) and patients in the intermediate NCCN risk group (100.0% vs 37.5%, P=0.001).3.The duration of agranulocytosis in the azacitidine combined with IA chemotherapy group was longer than that in the IA group (21 days vs 19 days, P=0.045). There was no significant difference in the number of platelet transfusions and the number of red blood cell transfusions between the two groups, and there was no significant difference in the incidence of infection between the two groups (table 2).Conclusions:The remission rate of induction chemotherapy for azacitidine combined with IA regimen and IA regimen in newly diagnosed non-M3 AML patients is comparable. Patients with a ratio of immature cells in bone marrow greater than 67% and patients in the intermediate NCCN risk group may benefit from azacitidine combined with the IA regimen. The combination of azacitidine with IA regimen aggravated granular bone marrow suppression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Genetic Expression Screening of Arsenic Trioxide-Induced Cytotoxicity in KG-1a Cells Based on Bioinformatics Technology.

Acute myeloid leukemia (AML) is a malignant tumor of the hematopoietic system, and leukemia stem cells are responsible for AML chemoresistance and relapse. KG-1a cell is considered a leukemia stem cell-enriched cell line, which is resistant to chemotherapy. Arsenic trioxide (ATO) is effective against acute promyelocytic leukemia as a first-line treatment agent, even as remission induction of relapsed cases. ATO has a cytotoxic effect on KG-1a cells, but the mechanism remains unclear. Our results demonstrated that ATO can inhibit cell proliferation, induce apoptosis, and arrest KG-1a cells in the G2/M phase. Using transcriptome analysis, we investigated the candidate target genes regulated by ATO in KG-1a cells. The expression profile analysis showed that the ATO had significantly changed gene expression related to proliferation, apoptosis, and cell cycle. Moreover, MYC, PCNA, and MCM7 were identified as crucial hub genes through protein–protein interaction network analysis; meanwhile, the expressions of them in both RNA and protein levels are down-regulated as confirmed by quantitative polymerase chain reaction and Western blot. Thus, our study suggests that ATO not only inhibits the expression of MYC, PCNA, and MCM7 but also leads to cell cycle arrest and apoptosis in KG-1a cells. Overall, this study provided reliable clues for improving the ATO efficacy in AML.

LSD1 Inhibition Exerts Its Anti-Leukemic Effect By Recommissioning PU.1- and C/Ebpα-Dependent Enhancers in AML

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. We aimed to investigate the chromatin changes induced by an irreversible inhibitor of LSD1 (GSK-LSD1) on rMLL+ leukemias. For this, we treated rMLL+ murine AML and patient-derived xenografts with the GSK-LSD1 inhibitor in vivo and assessed chromatin accessibility changes using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq). Treated mice exhibited a lower proportion of MLL-AF9+ leukemia cells detected in the bone marrow (BM) and signs of myeloid differentiation. Treated mice also had markedly improved survival with a median survival of 78 days vs. control mice (39 days). After 6 weeks of treatment with GSK-LSD1, mice xenotransplanted with human rMLL+ AML samples showed a significant decrease in engraftment and myeloid differentiation. Serial transplantation of murine AML revealed eradication of LSC upon GSK-LSD1 treatment of donor mice. Most importantly, LSD1 inhibition resulted in gain in chromatin accessibility in both murine and PDX-AML cells in vitro as well as in vivo . We then wanted to compare the effect of another anti-leukemic epigenetic drug, also known to induce myeloid differentiation of MLL-AF9+ cells, such as the DOT1L inhibitor EPZ4777. In contrast to GSK-LSD1, EPZ4777 treatment caused a global decrease in chromatin accessibility. The intersection of sites with dynamic accessibility upon DOT1L and LSD1 inhibition showed minimal overlap between the 2 treatments. The distribution of genomic regions with dynamic accessibility upon these drugs indicated that DOT1L inhibition affects a greater number of sites in promoter regions, whereas LSD1 inhibition affected a higher proportion of loci occupied within the intergenic and intronic regions. Furthermore, we sought to identify TF motif signatures at GSK-LSD1-induced dynamic sites. The most highly enriched motifs captured were those for myeloid TFs PU.1 and C/EBPα-β. We then performed ChIP-seq to identify sites of C/EBPα and PU.1 occupancy in the setting of GSK-LSD1 treatment. Interestingly, we observed a redistribution of PU.1 and C/EBPα occupancy upon treatment, consistent with the role of LSD1 in regulating the binding and activity of TFs. While PU.1 ChIPseq signal increased at dynamic ATAC-seq peaks in response to LSD1 inhibition, no increase in C/EBPα occupancy was observed. These dynamic sites also demonstrates GSK-LSD1-dependent co-occupancy of PU.1 and C/EBPα. We next sought to delineate whether PU.1 and C/EBPα are critical for these dynamic changes in chromatin accessibility in response to LSD1 inhibition. We performed ATAC-seq on PU.1 URE KO MLL-AF9 leukemias and C/EBPα KO MLL-AF9 leukemias treated with vehicle or GSK-LSD1. The increase in chromatin accessibility in TF-WT MLL-AF9 leukemias upon GSK-LSD1 was abrogated in both PU.1 URE KO and C/EBPα KO MLL-AF9 leukemia cells. We next assess the impact of TF-depletion on sensitivity to LSD1 inhibition. We treated PU.1 URE KO leukemias cells with GSK-LSD1 for 48 hours in vitro . These cells were more resistant to LSD1 inhibition when compared to PU.1 URE heterozygous and WT AML cells. In vivo, we reported loss of survival benefit upon GSK-LSD1 treatment in PU.1 URE KO MLL-AF9+ mice vs. WT mice. Similarly, C/EBPα KO leukemia cells were more resistant to LSD1 inhibition when compared to WT leukemia cells. After 2 weeks of treatment in vivo, C/EBPα KO leukemic mice also showed no decrease in MLL-AF9 allele burden. Taken together, our comparative investigations into the chromatin-modifying effects of two distinct epigenetic therapies targeting either LSD1 or DOT1L is illustrative of the concept that multiple paths toward myeloid differentiation can potentially be engaged to achieve therapeutic efficacy in AML. Our findings reveal that pharmacologic inhibition of LSD1 depends on balanced expression of the TFs PU.1 and C/EBPα, which can be used as predictors of response in AML aberrantly expressing these factors. DisclosuresMohammad:GSK: Employment. Ye:Incyte: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl:Celgene: Consultancy; Bayer Healthcare: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding. Kruger:GSK: Employment. Armstrong:Imago: Consultancy; Cyteir: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

DNA Demethylating Agents Generate a Brcaness Effect in Multiple Sporadic Tumor Types: Prediction for Sensitivity to PARP Inhibitors in AML

Poly (ADP-ribose) polymerase inhibitors (PARPis) have shown clinical efficacy in breast and ovarian cancers with inherited mutations in BRCA genes that abrogate homologous recombination (HR) DNA double-strand break (DSB) repair activity. However, to date, PARPishave not had success in sporadic breast and ovarian cancers, or other cancers, such as acute myeloid leukemia (AML), with intact BRCA genes. We have recently reported that triple-negative breast cancer (TNBC) and AML are sensitive to low doses of the potent PARPiTalazoparib in combination with DNA methyl transferase inhibitors (DNMTi), decitabine (DAC) or azacitidine (AZA), in vitro and in vivo. In these cancer models, the DNMTi/PARPicombination increases amplitude and retention of PARP1 directly at laser-induced DNA damage sites, increasing DNA DSBs, promoting synergistic tumor cytotoxicity, blunting cell self-renewal, and leading to strong anti-tumor responses. These results have led to a Phase I/II Clinical trial of DNMTi/PARPi combination therapy in patients with relapsed/refractory AML that is underway.In identifying the molecular pathways that may underlie efficacy of this inhibitor combination in sporadic TNBC, ovarian cancer and AML, we find that low doses of DNMTisreprogram the cancer genome, in part by changing gene expression patterns of DSB repair genes, potentially creating a HR defect (HRD) or “BRCAness” effect. Expression array (Agilent Human 44K v2) analysis was performed on RNA extracted from multiple BRCA-intact sporadic cell lines from AML (including, Kasumi-1, KG-1 and MOLM-14), TNBC (including, MDA-MB-231, SUM159) and ovarian cancer (including, A2780) on days 1, 3, 5 and 7 and 10 post DNMTi(DAC 10nM or AZA 500nM) treatment. Hallmark, KEGG and reactome gene ontologies were utilized. Only gene sets with log fold change (FDR) >.250 were considered significant and genes demonstrating 2-fold or greater change with drug treatment were selected. While multiple DNA repair pathway genes were found both up- and down- regulated post DNMTi treatment, one or more HR genes, were consistently downregulated in all cell lines examined. QPCR of mRNA and/or immunoblotting of HR proteins, in particular RAD51, BRCA1, BRCA2, or HR-related genes, FEN1 or FANCD2, showed significantly decreased expression with DNMTi treatment (p<0.05) in TNBC, ovarian and AML cell lines, validating array data. Functional assays in TNBC cell lines, using a stably expressed HR repair reporter (DRGFP) also confirmed decreased HR activity with DNMTi treatment. Additionally, in response to DNA damage with ionizing radiation (IR, 4Gy), RAD51 foci were reduced in AZA-treated TNBC and ovarian cancer cell lines, compared to untreated controls. Significantly increased triradial chromosomes were also observed in TNBC in the presence of AZA when exposed to DNA cross-linking agents mitomycin C (MMC, 50ng/ml) or cisplatin (2μM) treatment, confirming functional down-regulation of the HR-related FANCD2 pathway.The above HRD-like or BRCAness phenotype with DNMTi treatment translated to sensitivity to PARPis in primary bone marrow mononuclear cells (BMMNC) from AML patients. Thus, in 6 of 8 (75%) AML samples, HR gene expression by QPCR of RNA and HR functional activity by immunostaining for RAD51 foci, following DAC treatment (10nM, 72 hrs), DNMTi treatment resulted in a significant decrease in HR or HR-related gene expression, including RAD51, BRCA1, BRCA2, and decreased levels of RAD51 foci (p<0.05). All 6 AML samples were also significantly more sensitive to DAC/talazoparib combination treatment (p<0.05) in colony forming assays, compared with single agent treatments. AML samples included those with MLL translocations that are reported to have high HR activity and resistance to PARPis. Studies are underway to elucidate the molecular characteristics of the minority of AML patient samples (N=2) that did not generate a BRCAness effect with DAC treatment. In conclusion, our data suggest that one of the mechanisms underlying DNMTi/PARPi therapy efficacy in AML is DNMTi-induced BRCAness. In vitro testing for BRCAness with DNMTitreatment could be a biomarker to predict sensitivity to DNMTi/PARPis and inform selection of AML patients for future clinical trials of this inhibitor combination. DisclosuresCivin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; 3DBioWork Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kingsbury:3DBioWorks: Consultancy, Equity Ownership.

Interrogation of novel CDK2/9 inhibitor fadraciclib (CYC065) as a potential therapeutic approach for AML

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p&amp;lt;0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p&amp;lt;0.01 Ven+ or CD123-ENG T-cells versus control; p&amp;lt;0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents &amp; Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents &amp; Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; 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.

A Phase I/II Trial of CPX-351 + Palbociclib in Patients with Acute Myeloid Leukemia

A Phase I/II Trial of CPX-351 + Palbociclib in Patients with Acute Myeloid Leukemia

Co-delivery of homoharringtonine and doxorubicin boosts therapeutic efficacy of refractory acute myeloid leukemia

Refractory acute myeloid leukemia (AML) remains a challenging hematological malignancy to treat, due to the development of drug resistance, severe complications, and relapse in chemotherapies. Free-drugs combination has demonstrated enhanced therapeutic efficacy in AML, while it requires complicated administration regimens and brings added toxicity. To tackle this complex disease, in this work two clinically applied therapeutics, doxorubicin and homoharringtonine, were assembled into one polymeric micelle to form a co-delivery system (DHM) to facilitate a novel and simple administration regimen. The DHM was systematically investigated in the drug-resistant AML cell line HL60/A as well as in the AML1-ETO+-c-kit+ mouse featuring as a refractory and relapsed AML model following comprehensive characterizations. Compared with the free-drugs combination, DHM significantly enhanced the cellular uptake of the therapeutics, inhibited the cell division and induced a higher rate of cells apoptosis in vitro. More importantly, the intraperitoneal injection of DHM remarkably eradicated leukemia cells in the peripheral blood, bone marrow, spleen and liver of the AML mice and significantly prolonged the survival of the mice without additional systematic toxicity compared with that of the free-drugs combination. In conclusion, the DHM boosted the therapeutic effect of clinically applied chemodrugs as well as provided a novel platform for multi-drugs co-delivery against refractory and relapsed AML, therefore holding promising potential for translational medicine.

Biomarkers of Gemtuzumab Ozogamicin Response for Acute Myeloid Leukemia Treatment.

Gemtuzumab ozogamicin (GO, Mylotarg®) consists of a humanized CD33-targeted antibody-drug conjugated to a calicheamicin derivative. Growing evidence of GO efficacy in acute myeloid leukemia (AML), demonstrated by improved outcomes in CD33-positive AML patients across phase I to III clinical trials, led to the Food and Drug Administration (FDA) approval on 1 September 2017 in CD33-positive AML patients aged 2 years and older. Discrepancies in GO recipients outcome have raised significant efforts to characterize biomarkers predictive of GO response and have refined the subset of patients that may strongly benefit from GO. Among them, CD33 expression levels, favorable cytogenetics (t(8;21), inv(16)/t(16;16), t(15;17)) and molecular alterations, such as NPM1, FLT3-internal tandem duplications and other signaling mutations, represent well-known candidates. Additionally, in depth analyses including minimal residual disease monitoring, stemness expression (LSC17 score), mutations or single nucleotide polymorphisms in GO pathway genes (CD33, ABCB1) and molecular-derived scores, such as the recently set up CD33_PGx6_Score, represent promising markers to enhance GO response prediction and improve patient management.

Abstract A06: Impact of cigarette smoke exposure on acute myeloid leukemia progression

Abstract Cigarette smoke is known to cause lung cancer but is also associated with increased incidence of leukemia. Acute myeloid leukemia (AML) is the most lethal form of leukemia, and patients who are or were smokers have increased risk of developing AML and have worse prognosis. There is also an increased incidence of AML in children whose parents smoked either during or after the pregnancy. It is currently unknown how cigarette smoke exposure (CSE) may be impacting leukemic progression or treatment efficacy in AML. Cigarette smoking increases levels of reactive oxygen species (ROS) in the tissue of smokers, including peripheral blood samples. Oxidative stress can alter epigenetic signaling, including DNA methylation. Oxidation of methylated DNA and oxidative lesions on DNA can alter normal DNA methylation patterns. Smokers have altered DNA methylation that can be retained decades after the cessation of the habit, and these changes can be found in the blood cells of smokers. Cigarette smoke condensate (CSC) treatment has also been seen to epigenetically prime nontransformed human bronchial cells for malignant transformation. These occurrences have been seen in the context of lung cancer and lung damage, but studies into other tissue damage have largely been overlooked. Thus, we hypothesize that CSE impacts leukemic progression and treatment in AML through these mechanisms. We have developed a model to study the impact of CSE on AML in mice. NOD-SCID mice are smoke exposed for two weeks prior to being engrafted with luciferase-labeled human AML cells. The CSE is performed in the SCIREQ Cigarette Smoking Robot, with 3R4F research cigarettes 5 days/week. We include experimental arms of smoking cessation (who stop once evidence of leukemic engraftment is detected) and continued smoking, to model patients who either stop or continue smoking upon diagnosis of AML. Leukemic burden is quantified through bioluminescent imaging throughout the experiment. From these models, we have observed that two weeks of CSE is sufficient to significantly increase (p-value&amp;lt;0.0001) early leukemic burden as compared to non-smoke exposed mice, in FLT3-ITD AML-bearing mice. Spleen samples revealed trends of increased levels of superoxides in the CSE mice. Currently, immunohistochemistry is being performed on collected organs to detect oxidative stress markers. Additionally, mouse spleen DNA underwent reduced representative bisulfide sequencing; the levels of DNA methylation in the mouse host cells and in the leukemia are compared between our CSE groups. This is the first description of CSE promoting leukemia growth in mouse model, which will enable the study of mechanistic aspects. This may help define if unique treatment strategies are needed for these patients. Citation Format: Yue Lu, Marcos Estecio, Marina Konopleva, Elias Jabbour, Joya Chandra, Mary Figueroa. Impact of cigarette smoke exposure on acute myeloid leukemia progression [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A06.

MicroRNA-34a-mediated death of acute myeloid leukemia stem cells through apoptosis induction and exosome shedding inhibition via histone deacetylase 2 targeting.

We investigated the role of leukemia stem cells in chemoresistance and recurrence of acute myeloid leukemia. Total RNA was isolated from cells or tissues using TRIzol reagent. Cell viability was assessed with the tetrazolium assay. MicroRNA-34a (miR-34a), which acts on cell death regulation pathways, was noticeably downregulated in non-M3 acute myeloid leukemia stem cells compared with normal hematopoietic stem cells. Furthermore, inhibition of miR-34a-mediated suppression in leukemia stem cells was associated with poor clinical outcomes and impaired treatment efficacy in acute myeloid leukemia. Transfection with a miR-34a mimic triggered leukemia stem cell death and prevented leukemia. Bioinformatics analysis and a dual-luciferase reporter assay showed that miR-34a targeted the 3'-untranslated region of histone deacetylase 2, and the reinforced expression of miR-34a remarkably stimulated the expression of histone deacetylase 2 in leukemia stem cells. Ectopic miR-34a expression triggered death of leukemia stem cells via pathways involving the Janus kinase 1-signal transducer and activator of transcription 2-p53 axis. Targeting leukemia stem cells to trigger cell death through upregulation of miR-34a expression could be used to diagnose and treat acute myeloid leukemia.

Class I/IIb-Selective HDAC Inhibitor Exhibits Oral Bioavailability and Therapeutic Efficacy in Acute Myeloid Leukemia.

The HDAC inhibitor 4-tert-butyl-N-(4-(hydroxycarbamoyl)phenyl)benzamide (AES-350, 51) was identified as a promising preclinical candidate for the treatment of acute myeloid leukemia (AML), an aggressive malignancy with a meagre 24% 5-year survival rate. Through screening of low-molecular-weight analogues derived from the previously discovered novel HDAC inhibitor, AES-135, compound 51 demonstrated greater HDAC isoform selectivity, higher cytotoxicity in MV4-11 cells, an improved therapeutic window, and more efficient absorption through cellular and lipid membranes. Compound 51 also demonstrated improved oral bioavailability compared to SAHA in mouse models. A broad spectrum of experiments, including FACS, ELISA, and Western blotting, were performed to support our hypothesis that 51 dose-dependently triggers apoptosis in AML cells through HDAC inhibition.

Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Results of a Phase II Trial

Ten-Day Decitabine with Venetoclax (DEC10-VEN) in Acute Myeloid Leukemia: Updated Results of a Phase II Trial

Isoform Specific Regulation of Acute Myeloid Leukemia Maintenance By Wilms Tumor 1

The gene encoding Wilms Tumor 1 (WT1) is recurrently upregulated in Acute Myeloid Leukemia (AML). WT1 transcript burden is associated with primary refractory disease and with relapse when there is detectable WT1 minimal residual disease (Lambert et al., Oncotarget, 2014). We have recently described the gene regulatory networks in purified blasts from AML patients with various driver mutations (Assi et al., Nature Genetics, 2019) and found that WT1 was a key transcription factor node in all networks examined. Furthermore, in knockdown experiments as part of a shRNA depletion screen against transcription factors in vitro and in murine xenotransplantation experiments, we showed that WT1 was essential for leukemic maintenance (Martinez-Soria et al., Cancer Cell, 2018). WT1 produces 8 primary isoforms, all of which are expressed in hematopoietic cells by employing one of two transcription start sites and alternative splicing in exon 5 and exon 9. To further understand how WT1 maintains leukemia, we cloned each of the 8 isoforms into a doxycycline-inducible vector and transduced them into a t(8;21) AML cell line, Kasumi-1, which well recapitulates primary t(8;21) AML (Ptasinska et al., Leukemia, 2014). We carefully overexpressed each of the 8 WT1 isoforms to levels seen in AML patient samples and functionally and molecularly characterised them. Overexpression of isoforms lacking 3 amino acids in the zinc finger DNA binding domain (WT1 -KTS) significantly reduced cell growth and colony formation, increased apoptosis, caused a G1 cell cycle arrest and caused myeloid differentiation of leukemic blasts. However, overexpression of isoforms with the 3 amino acids (WT1 +KTS) in the DNA-binding domain had the opposite effects, thus having a major role in the maintenance of a leukemic phenotype. Also, we found that the expression of WT1 +KTS isoforms in purified blasts from patients was higher than WT1 -KTS isoforms and may therefore permit leukemic maintenance. We did not find any functional differences between isoforms employing alternate transcriptional start sites or between exon 5 splice variants. We then performed the first ever WT1 chromatin immunoprecipitation (ChIP-seq) in AML and show that WT1 -KTS shows increased binding as compared to WT1 +KTS with 9451 unique WT1 binding sites in WT1 -KTS expressing cells and only 1355 sites in WT1 +KTS expressing cells with 2391 sites overlapping between the isoforms. This finding suggests that WT1 isoforms compete for binding at multiple sites and may explain why the ratio of expression between WT1 isoforms is critical (Calabrese et al., Blood, 2012). We also correlated WT1 ChIP-seq peaks to transcription as assayed by RNA-seq. We found that differentially bound cis-regulatory elements are associated with altered expression of genes involved in proliferation, cell cycle, apoptosis and differentiation pathways, explaining many of the functional alterations in cellular behaviour that we see. Several vaccine studies and more recently a T cell receptor study targeted against WT1 have been undertaken but they have shown mixed efficacy in AML (Chapuis et al, Nature Medicine, 2019). Since we find that different isoforms of WT1 have antagonistic effects, we hypothesise that a more effective therapeutic strategy would be to selectively target only WT1 +KTS isoforms. Disclosures No relevant conflicts of interest to declare.

Outcomes of Relapsed or Refractory Acute Myeloid Leukemia after Frontline Hypomethylating Agent with Venetoclax Regimens

Outcomes of Relapsed or Refractory Acute Myeloid Leukemia after Frontline Hypomethylating Agent with Venetoclax Regimens

Aplastic Anemia induced by Nivolumab before a Treatment of Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

As an Immune checkpoint blockade therapy (ICB), nivolumab has demonstrated efficacy in Acute Myeloid Leukemia (AML) and various other malignancies. Nivolumab is used as an anti-programmed cell death 1 (PD-1) agent. The toxicities are observed in more than 10% of patients, because of its ability, anti-PD-1 will upregulate the activity of T-cells. Over-activated T-cells will cause immune-related adverse events such as Aplastic Anemia (AA). Here, we present a case of an over 60-years old male patient with AML, and the possibility for him to receive an allogeneic hematopoietic stem cell transplantation (allo-HSCT). The patient was treated with nivolumab and subsequently developed AA. As an additional consideration, we will also discuss whether allo-HSCT is transplantable when AA is performed during the treatment of AML.

Abstract 3949: Characterization of MP1115; a new natural killer cell activator

Abstract MP1115 is in development as a new immuno-oncology therapeutic lead comprised of bioactive phospholipid containing nanoparticles with activities unique to phospholipid active pharmaceutical ingredients. MP1115 demonstrated potent activation of NK cells in vitro and in vivo and had a secondary anti-tumor cell proliferation mechanism of action. Methods: MP1115 was formulated as ~100 nM diameter nanoparticles formulated in buffered sucrose and stably stored frozen. MP1115 nanoparticles were also loaded with a CDK8 inhibitor test compound at a 30 percent loading efficiency. MP1115 was tested for anti-tumor cell proliferation in 2D and 3D in vitro assays, NK cell activation in ex vivo splenocyte and NK cell activation assays, and anti-tumor efficacy and NK cell activation in vivo in immunocompetent and immunodeficient mice. MP1115-CDK8 inhibitor loaded nanoparticles were tested in vitro for increased anti-tumor cell proliferation compared to non-loaded MP1115 nanoparticles and non-loaded CDK8 inhibitor controls. Results: MP1115 demonstrated increased NK cell activation compared to a CDK8 test compound as measured by IFN-γ release in an ex vivo mouse splenocytes-Yac-1 mouse lymphoma assay (p&amp;lt;0.0001). This activity was maintained with splenocytes from immunocompetent BALB/c mice, immunodeficient BALB/c scid mice, and with purified NK cells from each strain background. MP1115 demonstrated increased NK cell activation of mouse splenocytes in vivo in immunocompetent BALB/c mice compared to controls (p&amp;lt;0.0001), as seen by increased CD69 and CD107a signal, as well as the significant killing of target Yac-1 cells ex vivo (p&amp;lt;0.001). The levels of activation and killing observed were similar to that achieved by a CDK8 inhibitor test compound (p&amp;gt;0.9999). MP1115 also successfully demonstrated the ability to deliver a CDK8 test compound payload to human non-Hodgkin’s lymphoma cells in vitro with increased anti-tumor cell proliferation compared to controls (p&amp;lt;0.0001). MP1115 achieved increased anti-tumor cell proliferation efficacy in acute myeloid leukemia, multiple myeloma, non-Hodgkin’s lymphoma, non-small cell lung cancer, KRAS mutant colorectal cancer models in vitro, and in pancreatic cancer tumor models in vivo. MP1115 demonstrated high tolerability in rodent models when dosed at over 200 mg/kg daily for multiple doses. MP1115 is currently being validated in additional in vivo models including human immune system mice with human xenografts. Conclusions: MP1115 is being advanced to IND-enabling studies based on demonstrated low toxicity, high systemic delivery, potent NK cell activation, and anti-tumor cell proliferation activities. Citation Format: Pitchaimani Kandasamy, Jason E. Duex, Greg Miknis, Julie Lang, Ana Chauca-Diaz, Chris Johnson, Colleen Hudson, Jakub Staszak-Jirkovsky, Neal C. Goodwin. Characterization of MP1115; a new natural killer cell activator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3949.