Chemotherapy-resistant Acute Myeloid Leukemia Research Articles

ALDH3A2 Regulates Ferroptosis By Participating in Fatty Acid Synthesis and Finally Lead to Chemotherapy Resistance in Acute Myeloid Leukemia

Chemotherapeutic resistance is a major challenge in the treatment of acute myeloid leukemia (AML). Although increasing the dose of anthracycline drugs improve chemotherapy efficacy and reduce drug resistance, the side effects limit the increase of their dosage. One strategy to improve the therapeutic effects is to improve the efficacy in their limited doses. Ferroptosis is one important way in which Adriamycin exerts cytotoxic effects on AML cells, which was also associated with resistance to Adriamycin. ALDH3A2 is a fatty aldehyde dehydrogenase related to the conversion of fatty aldehydes and fatty acids. Here, we found that ALDH3A2 is a biomarker for chemotherapy-resistant AML, increased expression of ALDH3A2 reduced ferroptosis and cytotoxicity induced by Adriamycin in AML cells. In our studies, we demonstrated that ALDH3A2 regulated ferroptosis and AML drug resistance by participating in fatty acid synthesis. By analyzing the data from TCGA and GTEx databases, we found that ALDH3A2 expression varied in different types of tumor cells. AML patients had higher ALDH3A2 expression than normal tissues. We analyzed the RNA-seq results of 88 leukemia cell lines from the HPA database, and found that 15 out of the top 25% highest ALDH3A2 expression cell lines were AML cell lines, indicating that AML cells had higher ALDH3A2 expression than other types of leukemia cells. We plotted survival curves based on the gene data and clinical survival data from TCGA and GTEx databases. It showed that AML patients with high ALDH3A2 expression had shorter overall survival (OS) than those with low expression. We detected the expression of ALDH3A2 in AML cell lines and primary cells from patients using Western blot and RT-qPCR, respectively. Our data showed that drug-resistant AML cell lines had higher ALDH3A2 expression than their parental cells. The expression of ALDH3A2 in primary cells from AML patients achieving continuous complete remission (CR) within two years was significantly lower than that in the first-line non-remission(C1NR) group and the relapsed/refractory (R/R) group. Besides, the expression of ALDH3A2 is related to certain types of mutation such as CEBPα, FLT3-ITD and RUNX1. To investigate the function of ALDH3A2, we constructed AML cell lines with ALDH3A2 knockdown or overexpression. We found that the increasing ferroptosis level and decreasing cell activity after treatment with the same concentration of Adriamycin was observed in the knockdown group, and overexpression group had the opposite effect. By ELISA and GC-MS, we discovered that there were differences in 4-HNE and fatty acid content between the overexpression group and the control group. It suggested that ALDH3A2 overexpression reduced the cytotoxic 4-HNE and increased the content of heptadecanoic acid, oleic acid, and linoleic acid. We further pretreated AML cells with heptadecanoic acid, oleic acid, and linoleic acid, and found that pretreatment with heptadecanoic acid and oleic acid reduced ferroptosis level and cytotoxicity induced by Adriamycin in AML cells. We also tried to find the regulatory mechanism of ALDH3A2 expression and found that acetylation plays a certain role in it. In conclusion, our studies found that increased expression of ALDH3A2 was related to poor chemotherapy efficacy in AML patients and clarifies the mechanism of ALDH3A2 regulating chemosensitivity in AML. ALDH3A2 degraded the cell toxic product 4-HNE of lipid peroxidation and regulated the proportion and content of heptadecanoic acid, oleic acid, and linoleic acid in cells, which related to ferroptosis and resistance to anthracycline drugs. Downregulating ALDH3A2 could increase AML's susceptibility to chemotherapy which may make it a promising therapy.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR01.

Chemotherapeutic drugs elicit stemness and metabolic alteration to mediate acquired drug-resistant phenotype in acute myeloid leukemia cell lines

Chemotherapy resistance leading to disease relapse is a significant barrier in treating acute myeloid leukemia (AML). Metabolic adaptations have been shown to contribute to therapy resistance. However, little is known about whether specific therapies cause specific metabolic changes. We established cytarabine-resistant (AraC-R) and Arsenic trioxide-resistant (ATO-R) AML cell lines, displaying distinct cell surface expression and cytogenetic abnormalities. Transcriptomic analysis revealed a significant difference in the expression profiles of ATO-R and AraC-R cells. Geneset enrichment analysis showed AraC-R cells rely on OXPHOS, while ATO-R cells on glycolysis. ATO-R cells were also enriched for stemness gene signatures, whereas AraC-R cells were not. The mito stress and glycolytic stress tests confirmed these findings. The distinct metabolic adaptation of AraC-R cells increased sensitivity to the OXPHOS inhibitor venetoclax. Cytarabine resistance was circumvented in AraC-R cells by combining Ven and AraC. In vivo, ATO-R cells showed increased repopulating potential, leading to aggressive leukemia compared to the parental and AraC-R. Overall, our study shows that different therapies can cause different metabolic changes and that these metabolic dependencies can be used to target chemotherapy-resistant AML.

Targeting cell cycle and apoptosis to overcome chemotherapy resistance in acute myeloid leukemia.

Chemotherapy-resistant acute myeloid leukemia (AML), frequently driven by clonal evolution, has a dismal prognosis. A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockouts involving AraC metabolism and genes that regulate cell cycle arrest (cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53) as contributing to resistance. In human AML cohorts, reduced expression of CDKN2A conferred inferior overall survival and CDKN2A downregulation occurred at relapse in paired diagnosis-relapse samples, validating its clinical relevance. Therapeutically targeting the G1S cell cycle restriction point (with CDK4/6 inhibitor, palbociclib and KAT6A inhibitor, WM-1119, to upregulate CDKN2A) synergized with chemotherapy. Additionally, direct promotion of apoptosis with venetoclax, showed substantial synergy with chemotherapy, overcoming resistance mediated by impaired cell cycle arrest. Altogether, we identify defective cell cycle arrest as a clinically relevant contributor to chemoresistance and identify rationally designed therapeutic combinations that enhance response in AML, potentially circumventing chemoresistance.

Upregulated mitosis-associated genes CENPE, CENPF, and DLGAP5 predict poor prognosis and chemotherapy resistance of Acute Myeloid Leukemia.

Mitosis-associated genes are dysregulated in many types of cancers and play important roles in disease progression and chemotherapy resistance. However, their expression and functions in chemotherapy-resistant Acute Myeloid Leukemia (AML) are still largely undetermined. This study aims to explore the roles of spindle assembly checkpoint (SAC) genes CENPE, CENPF, and DLGAP5 in chemotherapy-resistant AML. RNA-sequencing (RNA-seq) was performed in patients with chemotherapy-resistant AML and chemotherapy-sensitive AML. AML mRNA data from 151 patients with recurrence were downloaded from TCGA. Integrated analysis of the differentially expressed genes (DEGs), GO and KEGG pathways. CENPE, CENPF, or DLGAP5 knockdown cell lines were used to analyse proliferation, apoptosis and cell cycle alterations. A total of 87 DEGs (48 upregulated and 39 downregulated) were obtained through gene analysis of R/R-AML and a total of 329 DEGs (202 upregulated and 127 downregulated) were obtained in refractory S-AML. Upregulated DEGs were mainly enriched in cell cycle (GO: 0007049, hsa04110) and mitotic cell cycle (GO: 0000278) processes and pathway. Venn diagram analysis identified the most upregulated DEGs (including CENPE, CENPF, and DLGAP5) in chemoresistant AML. The expression of CENPE, CENPF and DLGAP5 in R-AML (TCGA) was significantly higher than that of primary AML (GEO). The proliferation of K562 cells after CENPE and DLGAP5 knockdown was significantly decreased (P= 0.0001 and P= 0.0006). In THP-1 cells, the CCK-8 values after CENPE, CENPF and DLGAP5 knockdown were significantly decreased (P= 0.01, P= 0.0395 and P= 0.0362). Knockdown of CENPE, CENPF and DLGAP5 significantly increased cell apoptosis by regulating Caspase-9, BAX, TP-53 and bcl-2, and induced cell cycle arrested by regulating CDK1, CDK2, CDKN1A, and CyclinD1. In conclusion, the mitotic cell cycle-associated genes CENPE, CENPF, and DLGAP5 were upregulated in chemotherapy-resistant AML patients and might be useful for predicting poor prognosis.

Targeting histone methylation to reprogram the transcriptional state that drives survival of drug-tolerant myeloid leukemia persisters

SummaryAlthough chemotherapy induces complete remission in the majority of acute myeloid leukemia (AML) patients, many face a relapse. This relapse is caused by survival of chemotherapy-resistant leukemia (stem) cells (measurable residual disease; MRD). Here, we demonstrate that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing histone 3 lysine 27 (H3K27) and H3K4 tri-methylation. Within a doxorubicin-sensitive leukemia cell population, we identified a subpopulation of reversible anthracycline-tolerant cells (ATCs) with leukemic stem cell (LSC) features lacking doxorubicin-induced H3K27me3 or H3K4me3 upregulation. These ATCs have a distinct transcriptional landscape than the leukemia bulk and could be eradicated by KDM6 inhibition. In primary AML, reprogramming the transcriptional state by targeting KDM6 reduced MRD load and survival of LSCs residing within MRD, and enhanced chemotherapy response in vivo. Our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of transcriptional reprogramming by epigenetic-based therapeutics to target chemotherapy-resistant AML cells.

Opioid receptor signaling suppresses leukemia through both catalytic and non-catalytic functions of TET2.

Acute myeloid leukemia (AML) is a genetically heterogeneous and frequently fatal malignancy. The ten-eleven translocation (TET)-mediated DNA demethylation is known to be critically associated with AML pathogenesis. Through chemical compound screening, we find that the opioid receptor agonist, loperamide hydrochloride (OPA1), significantly suppresses AML cell viability. The potential therapeutic effects of opioid receptor agonists, especially OPA1, are verified in AML cells invitro and mouse and human AML models invivo. OPA1-induced activation of OPRM1 signaling enhances the transcription of TET2 and thus activates both catalytic-dependent and -independent functions of TET2. Notably, AMLs with TET2 mutations or chemotherapy resistance are sensitive to OPA1 as well. Our results reveal the OPRM1-TET2 regulatory axis in AML and suggest that opioid agonists, particularly OPA1, a US Food and Drug Administration (FDA)-approved antidiarrheal drug, have therapeutic potential in AML, especially in TET2-mutated and chemotherapy-resistant AMLs, which have a poor prognosis.

Targeting Control of Cell Cycle Enhances the Activity of Conventional Chemotherapy in Chemotherapy-Resistant Acute Myeloid Leukemia

Chemotherapy-resistant acute myeloid leukemia (AML) manifesting as primary refractory or relapsed disease carries a dismal prognosis and is often driven by clonal evolution. We have performed a genome-wide CRISPR knockout screen investigating resistance to conventional AML therapy, cytarabine and doxorubicin (AraC/Dox), in 2 independent human AML cell lines. Chemoresistant populations were enriched with gRNAs disrupting cell cycle arrest genes, including cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53. Here, we validate the direct contribution of these genes to chemoresistance and demonstrate that rationally designed therapeutic regimens targeting cell cycle enhances chemotherapy response in AML.Using CRISPR-mediated gene editing, we individually inactivated CDKN2A and CHEK2 in Cas9-expressing OCI-AML3 and MV4-11 cells. CDKN2A- and CHEK2-deficient cells demonstrated proliferative advantage in the presence of AraC/Dox in co-culture competition assays, confirming direct contribution of these gene knockouts to chemoresistance. Nil to modest reductions in apoptosis were seen in CDKN2A- and CHEK2-deficient cells treated with AraC/Dox for 72 hours compared to unedited controls. However, failure of accumulation of cells in the non-cycling G 0/G 1 proportion was seen in these edited cells after chemotherapy, corresponding with maintained DNA synthesis, as measured by BrdU incorporation, and a failure to downregulate phospho-Rb protein expression, indicating ongoing active cell cycling in spite of chemotherapy. These results confirm failure of cell cycle arrest as the major mechanism of resistance with inactivation of CDKN2A or CHEK2.To assess relevance of these genes in chemotherapy response in human AML, we analysed the effect of CDKN2A expression on prognosis. Reduced expression of CDKN2A conferred inferior overall survival in 3 independent clinical cohorts. Additionally, downregulation of CDKN2A and an increase in downstream cell cycling effector, cyclin-dependent kinase 6 (CDK6) was seen at relapse in paired diagnosis-relapse AML samples (Li et al. 2016, Nature Medicine). Further, CHEK2 mutations in clonal hematopoiesis are enriched in patients with solid organ cancers following chemo- or radiotherapy, functionally demonstrating chemoresistance over non-mutated cells. These data suggest CDKN2A and CHEK2 loss-of-function is relevant in promoting chemoresistance in human hematopoiesis.We therefore investigated whether therapeutically targeting cell cycle control pathways that converge on the G 1/S restriction point could synergise with cytotoxic chemotherapy, potentially circumventing chemoresistance. The addition of MDM2 inhibitor, nutlin-3a with AraC/Dox achieved striking synergism in TP53-competent cell lines in reducing viability and promoting apoptosis. Inhibition of CDK4/6 with palbociclib alone induced cell cycle arrest without apoptosis, however combination therapy with AraC/Dox significantly potentiated apoptosis. To functionally validate the role of CDKN2A itself, we examined the inhibitor of histone acetyltransferase KAT6A, WM-1119 (Baell et al. 2018, Nature), which transcriptionally upregulates CDKN2A. WM-1119 had anti-leukemic activity across multiple cell lines, corresponding with down regulation of cell cycling and upregulation of senescence signatures. The anti-leukemic activity was prevented by CDKN2A inactivation, indicating that CDKN2A upregulation is essential for WM-1119's anti-leukemic effect. WM-1119 enhanced activity of AraC/Dox in multiple cell contexts, mediated by cooperative induction of CDKN2A. Its efficacy and synergy with AraC/Dox was further demonstrated in MLL-AF9 primary murine AML ex vivo, demonstrating broader applicability of the approach.Our findings reveal defects in cell cycle arrest pathways as clinically relevant contributors to chemoresistance in AML. Combining agents to target cell cycle components improved in vitro anti-leukemic activity of chemotherapy and warrants further exploration of their translational potential. DisclosuresBullinger: Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Menarini: Consultancy; Hexal: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Astellas: Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Seattle Genetics: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Consultancy, Honoraria. Lane: BMS: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy; Geron: Consultancy.

The CD226-ERK1/2-LAMP1 pathway is an important mechanism for Vγ9Vδ2 T cell cytotoxicity against chemotherapy-resistant acute myeloid leukemia blasts and leukemia stem cells.

Vγ9Vδ2 T cells are attractive effector cells for immunotherapy with potent cytotoxic activity against a variety of malignant cells. However, the effect of Vγ9Vδ2 T cells on chemotherapy‐resistant acute myeloid leukemia (AML) blasts, especially highly refractory leukemia stem cells (LSCs) is still unknown. In this study, we investigated the effect of cytotoxicity of allogeneic Vγ9Vδ2 T cells on chemotherapy‐resistant AML cell lines, as well as on primary AML blasts and LSCs obtained from refractory AML patients. The results indicated that Vγ9Vδ2 T cells can efficiently kill drug‐resistant AML cell lines in vitro and in vivo, and the sensitivity of AML cells to Vγ9Vδ2 T cell–mediated cytotoxicity is not influenced by the sensitivity of AML cells to chemotherapy. We further found that Vγ9Vδ2 T cells exhibited a comparable effect of cytotoxicity against LSCs to primary AML blasts. More importantly, we revealed that the CD226–extracellular signal–regulatory kinase1/2 (ERK1/2)–lysosome‐associated membrane protein 1 (LAMP1) pathway is an important mechanism for Vγ9Vδ2 T cell–induced cytotoxicity against AML cells. First, Vγ9Vδ2 T cells recognized AML cells by receptor‐ligand interaction of CD226–Nectin‐2, which then induced ERK1/2 phosphorylation in Vγ9Vδ2 T cells. Finally, triggering the movement of lytic granules toward AML cells induced cytolysis of AML cells. The expression level of Nectin‐2 may be used as a novel marker to predict the susceptibility/resistance of AML cells to Vγ9Vδ2 T cell treatment.

Chemotherapy-Refractory Acute Myeloid Leukemia and Outcomes after Allogeneic Stem Cell Transplantation—a Single Center Analysis

For patients with acute myeloid leukemia (AML) who fail to achieve complete remission (CR) after induction chemotherapy, allogeneic stem cell transplantation (Allo-SCT) can provide leukemia control and result in long-term survival (Blood. 2016;127(1):53-61; BBMT. 2003;9(12):766-71; BBMT. 2016;22(10):1867-73). In this retrospective study, we analyzed the outcomes of patients at our institution who had chemotherapy-resistant AML and subsequently received Allo-SCT. We defined “chemotherapy-resistant AML” as not achieving CR prior to transplant according to the Center for International Blood & Marrow Transplant Research (CIBMTR) reporting criteria after one or more cycles of induction chemotherapy. We queried our CIBMTR reporting data and identified 44 patients age ≥ 18 years with AML who were not in CR and who were transplanted from 2009 to 2015. Patient records were reviewed and the following variables were collected: age, KPS, cytogenetic risk, number of prior chemotherapy cycles, chemotherapy regimens, transplant conditioning regimens, stem cell donor characteristics, blasts percentage at time of transplantation, and key transplant outcomes (overall survival, OS; relapse mortality; and, non-relapse mortality, NRM). Key transplant outcomes were segregated by blast percentage at time of transplantation (≤5% v. >5%) and analyzed. The median age was 60 years (range 20-73 years), and the median KPS was 62%(range 50-100%). Most patients received 2 or more induction regimens prior to transplant (n=32, 72%). Based on the MRC 2010 cytogenetic risk classification system (Blood. 2010 116:354-365), cytogenetic risk was classified as intermediate risk in 23 patients and unfavorable risk in 21 patients. Molecular markers and targeted gene sequencing were not available in most cases. Myeloablative conditioning was used in 29 patients (65%). Stem cell donors were as follows: matched related donor (n= 6; 13.9%), 10/10 or 9/10 matched unrelated donor (n=25, 56%), haploidentical donor (n= 6; 13.9%), and cord blood donor (n= 6; 13.9%). Stem cell sources were as follows: peripheral blood (n= 34; 79%), bone marrow (n= 3, 6.9%), and cord blood (n= 6; 13.9%). Blast count at the time of transplant ranged from <5% to as high as 55%; 18 patients (41%) had blast count >5% at the time of transplant. With a median follow up of 553days, cumulative 1-year and 2-year survival for the entire cohort was 49.5% and 37.8%, respectively. Most of the deaths occurred within the first year after the transplant (n=21; 49%). Of the 30 deaths, relapse preceded death in 17 patients (57%). A graphical representation of outcomes (swimmer plot) summarizes the post-transplant outcome for each of the 44 patients (Figure 1). Our data suggest that elevated blast percentage pre-transplant may impact survival outcomes in patients with chemotherapy-resistant AML, through the results were not statistically significant (Figure 1; ≤5% blasts median OS 638 days v. >5% 228 days; p=0.14). Approximately equal numbers of patients died from disease relapse and NRM in this high-risk cohort regardless of the blast percentage prior to transplantation (Figure 1). A third of patients are currently alive and disease free (n=14; 32%), with relatively equal numbers in the ≤5% blast and >5% blast groups (n=8 v. n=6). Although our study involves a small population of patients, we conclude that the approach to this high-risk subgroup of patients requires further study and a more standardized approach. Strategies to reduce non-relapse mortality (improved conditioning regimens) and relapse mortality (post-transplant maintenance strategies) are equally important in this group who otherwise have few treatment options. [Display omitted] DisclosuresLin:Jazz Pharmaceuticals: Consultancy. Ganguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau. Skikne:Celgene Corporation: Employment, Equity Ownership.

Low Expression of GFI-1 Gene Is Associated with Chemotherapy-Resistant in Acute Myeloid Leukemia through Influencing the Level of HO-1

In order to improve the poor cure rate of acute myeloid leukemia (AML), epigenetic modification has become a new treatment of leukemia in recent years. As a zinc-finger transcriptional repressor exerts, GFI-1 were found associated with poor prognosis in AML, but the underlying mechanism is not clear. Our investigation committed to studying the potential mechanism of resistance to Panobinostant in AML with low expression of GFI-1. Low level of GFI-1 implicated the poor prognosis of AML patients. In this study, we investigated the correlation of GFI-1 at transcription level in de novo AML patients. Follow up detection the progress of treatment of patients. After two courses of induction, we selected 32 patients in complete remission(CR stages)and 32 patients in relapsed or drug-resistant (relapsed stages), samples were selected to collect at the time of initial diagnosis. The transcription level of GFI-1 found that low expression of Gfi-1 also leads to chemotherapy resistance(P<0.05). Then our study explored its mechanisms of chemoresistance to Panobinostant invitro. Same with Same result in the vitro experiment, GFI-1 knock down by siRNA eliminated Panobinostat -induced cell apoptosis. Meanwhile, low level GFI-1 could enhance HO-1 expression at mRNA and protein level. As a previous study in our laboratory, HO-1 high expression could improve the sensitivity of drug resistant and tumor prolife in AML. However, correlation between GFI-1 and HO-1 has been no reported. So we try to found correlation between GFI-1 and HO-1 on chemotherapeutic response and explored the underlying mechanism. We regulated the level of HO-1 and found that the apoptosis rate between the low level of GFI-1 and empty vector has eased. GFI-1 regulates the HO-1 through the PI3K-AKT pathway, which affects the sensitivity of the cells to Panobinostat .Our data show that HO-1 plays a crucial role in GFI-1-dependent in chemotherapy-resistant AML. This provides a novel promising candidate for myeloid leukemia therapeutic method. DisclosuresNo relevant conflicts of interest to declare.

Targeting Residual Chemotherapy-Resistant Acute Myeloid Leukemia Cells By a Novel Oxphos Inhibitor IACS010759

Acute myeloid leukemia (AML) is initiated and maintained by a relatively rare leukemia stem cells (LSCs) that are capable of self-renewing, proliferation and differentiation into leukemia blasts. Recent data showed that LSCs (Lagadinou et al. Cell Stem Cell 2013) and residual cytarabine (Ara-C)-resistant AML cells (Farge et al. Cancer Discovery 2017) are highly dependent on mitochondrial function for survival. This unique metabolic biology makes LSCs and AML cells resistant to chemotherapy vulnerable to oxidative phosphorylation (OXPHOS) inhibition. We have identified a novel potent inhibitor, IACS-010759, which blocks mitochondrial complex I, suppresses OXPHOS and selectively inhibits the growth of AML cells (Molina et al. AACR 2016). In this study, we aimed to determine the effects of OXPHOS inhibition with IACS-010759 on residual AML cells and AML LSCs surviving standard chemotherapy (Doxorubicin/Ara-C, DA) in patient-derived xenograft (PDX) AML models.Consistent with Farge et al study, OCI-AML3 cells treated with DA in vitro produced elevated levels of reactive oxygen species, displayed increased mitochondrial membrane potential and higher mitochondrial mass (data not shown), indicating reliance on mitochondrial metabolism. Further, Ara-C facilitated statistically significant increase in the basal and maximal oxygen consumption rates (OCR) (36%±8%, p=0.03; 36%±3%, p=0.003, respectively) compared to control. In turn, OXPHOS inhibition with IACS-010759 at 30 nM fully inhibited basal and Ara-C-induced OCR (Fig. 1A). These findings indicate that chemotherapy fosters mitochondrial respiration in AML, which could be abrogated by OXPHOS inhibitor.To examine the efficacy of IACS-010759 on residual leukemia cells surviving chemotherapy in vivo, we first established a chemosensitive PDX AML model of minimal residual disease. Treatment of mice inoculated with a human AML PDX harboring FLT3-ITD mutation with DA (D: 1.5 mg/kg; A: 50 mg/kg) reduced leukemia burden (PB, 0.8 ± 0.6% vs 45.8 ± 8.2% in vehicle-treated mice, p=0.001). The residual AML cells in DA-treated mice expanded and caused rapidly progressing leukemia (78.2 ± 6.2% vs 95.3 ± 1.0% in vehicle-treated mice, p=0.047) on week 6 post DA. Daily oral treatment of mice with IACS-010759 (7.5 mg/kg) as a single agent reduced leukemia burden, and caused further reduction when administered post completion of DA (Fig. 1B,C). The CyTOF mass cytometry, which enables measurement of up to 40 parameters per cell, demonstrated reduced frequency of CD34+CD38lowCD123+ AML LSCs and resurgence of CD11c+ differentiated cells in both IACS and IACS/DA groups (Fig. 1D). On the contrary, chemotherapy alone failed to significantly reduce fractions of LSCs or induce differentiation (Fig. 1D). Proliferation measured by Ki67 was greatly reduced by IACS/DA combination in all populations (1.4 ± 0.3% vs 5.5 ± 0.4% in vehicle group, p<0.01). The expression of hypoxia-inducible factor 1α was downregulated, consistent with the decreased oxygen consumption by IACS-010759 (not shown).We next investigated the efficacy of IACS-010759 in a chemoresistant PDX model inoculated with chemorefractory MLL-rearranged AML. CyTOF analysis demonstrated that DA chemotherapy failed to reduce the primitive leukemia stem/progenitor populations, which comprised 84.7% of total cells in DA versus 71.2% in vehicle groups, while the combination of IACS-010759 and DA efficiently reduced this population down to 30.2%. Additionally, the combination increased the proportion of mature cells characterized by CD34-CD64+CD11b+ (43.1% versus 12.8% in vehicle), which remained low in the DA-only group (5.6%). DA/OXPHOS inhibitor combination significantly extended survival, compared to the vehicle or single-agent treatment arms (Fig. 1E).In conclusion, AML cells surviving chemotherapy depend on OXPHOS for survival, and OXPHOS inhibition with complex I inhibitor IACS-010759 reduces AML LSC pool, induces differentiation and extends survival in AML PDX models in vivo . Our data advocate for combining IACS-010759 with standard chemotherapy, upon identification of a recommended Phase II dose in an ongoing clinical trial of IACS-010759 in AML (NCT02882321). [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding. Andreeff:Daiichi Sankyo: Consultancy. Kantarjian:ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding. Daver:Karyopharm: Consultancy, Research Funding; Incyte Corporation: Honoraria, Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Jazz: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Pfizer Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Kiromic: Research Funding; Immunogen: Research Funding.

Abstract PO-125: Targeting histone methylation to overrule transcriptionally driven drug resistance in myeloid leukemia

Abstract Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapeutic to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions (CR) in the majority of patients, many face a relapse. Relapse is caused by the survival of chemotherapy-resistant leukemia cells (minimal residual disease, MRD), that may have acquired drug resistance by non-genetic mechanisms. Persistence of MRD is driven by intra-leukemic heterogeneity and plasticity in response to therapy. Leukemia cells within MRD capable of initiating relapse are thought to have stem cell features and are therefore named “leukemic stem cells” (LSCs). In this study, we demonstrated that the anthracycline doxorubicin epigenetically reprograms leukemia cells by inducing tri-methylation of histone 3 lysine 27 (H3K27) and H3K4. Moreover, to understand chemotherapy resistance at the single-cell level and to study intra-leukemia heterogeneity with respect to anthracycline sensitivity, we seeded K562 myeloid leukemia cells in 480 wells, each containing 10.000 cells, and treated the cells with increasing concentrations of doxorubicin. Using this approach, a subpopulation of reversible anthracycline-tolerant cells (ATCs) was identified within the doxorubicin-sensitive leukemia cell population, which lacked upregulation of H3K27me3 or H3K4me3 and exhibited epigenetically regulated expression of stem cell features similar to leukemic stem cells (LSCs). These ATCs have a distinct transcriptional landscape from the leukemia bulk and could be eradicated by the KDM6 inhibitor GSK-J4. In primary human AML, GSK-J4 treatment could eliminate AML blasts and LSCs at diagnosis but importantly it could significantly reduce chemotherapy resistant AML patient cell load (MRD) and survival of LSCs residing within MRD, ex vivo and in vivo. An efficient response to GSK-J4 was associated with upregulation of H3K27 and/or H3K4 methylation, and downregulation of STAT5B. Together, our results reveal plasticity of anthracycline resistance in AML cells and highlight the potential of epigenetic-based therapeutics to target chemotherapy-resistant AML cells. Citation Format: Noortje van Gils, Han J.M.P. Verhagen, Tània Martiáñez, Fedor Denkers, Eline Vermue, Arjo Rutten, Tamás Csikós, Meyrem Çil, Marjon Al, Frank van Gemert, Jeroen J.W.M. Janssen, Gert J. Ossenkoppele, Renee X. Menezes, Linda Smit. Targeting histone methylation to overrule transcriptionally driven drug resistance in myeloid leukemia [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-125.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation

AbstractAtovaquone, a US Food and Drug Administration–approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Suppressing Hedgehog signaling reverses drug resistance of refractory acute myeloid leukemia.

BackgroundHedgehog (Hh) signaling is involved in the pathogenesis of tumors. By performing gene chip analysis, we predicted that Hh signaling might regulate multiple downstream pathways in acute myeloid leukemia (AML).MethodsIn this study, the potential role of the Hh pathway in refractory AML, and the impact of Hh expression on clinical prognosis were examined. We also investigated the role of the Hh inhibitor NVP-LDE225 in reversing drug resistance of refractory primary AML cells in vitro and the roles of multiple drug-resistant HL60/Adriamycin-resistant cells in vitro and in vivo (in a xenograft mouse model). Finally, we explored the underlying mechanisms.ResultsHh pathway was highly active in chemotherapy-resistant AML cells; by contrast, activation was less pronounced in chemosensitive cells and non-refractory primary cells. Strong activation of this pathway was associated with higher recurrence rates and poorer relapse-free and overall survival. NVP-LDE225 inhibited MRP1 protein expression, increased intracellular accumulation of Adriamycin, and reversed chemotherapeutic resistance. These effects were likely mediated through inhibition of the IGF-1R/Akt/MRP1 pathway. In the AML xenograft mouse model, NVP-LDE225 plus Adriamycin resulted in marked tumor regression.ConclusionThese findings suggest that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance and preventing refractory AML.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia

To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects invitro and invivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.

Potential CD34 signaling through phosphorylated-BAD in chemotherapy-resistant acute myeloid leukemia

Acute myeloid leukemia (AML) constitutively express growth factors and cytokines for survival. Chemotherapy alters these signals to induce cell death. However, drug resistance in AML remains a major hindrance to successful treatment and early warning is unavailable. Modulation of signaling pathways during chemotherapy may provide a window to detect response and predict treatment outcome. Blood samples collected from AML patients before and at day-3 of induction therapy were compared for changes in expression of CD117, CD34, pro-inflammatory cytokines and mediators of Akt and MAPK pathways, using multi-color flow cytometry. Nine patients were diagnosed as drug-resistant and seven sensitive to chemotherapy. Twelve were paired. Average percentages of CD34 (66.8 ± 11.7% vs. 26.2 ± 5.8%, p = 0.033) and pBAD (66.9 ± 8.2% vs. 28.9 ± 8.2%, p = 0.016) were significantly increased in chemo-resistant (N = 9) compared to chemo-sensitive (N = 5) samples. Percentages of CD34 were strongly correlated with pBAD (R = 0.785; p = 0.001; N = 14) and pFKHR (R = 0.755; p = 0.002; N = 14) at day-3 induction. Chemo-sensitive cases expressed significantly higher percentages of IL-18Rα (71.9 ± 9.6% vs. 29.8 ± 5.8%, p = 0.016). Though not significantly different in the outcome, IL-1β was strongly associated with activated Akt-S473, IL-6 with phosphorylated JNK and FKHR while TNF-α appeared to trigger Bim, in treated samples. These preliminary results suggested AML cells resistant to chemotherapy increased expression of CD34 and may signal through pBAD while cells sensitive to chemotherapy-induced IL18Rα expression. These were observed early during induction therapy. Identifying CD34 is interesting as it is a convenient marker to monitor drug-resistance in AML patients. Inhibition of CD34 and pBAD signaling may be important in treating drug-resistant AML.

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy

BackgroundWhile conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease.MethodsDrug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity.ResultsHere, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity.ConclusionsOur results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

VEGFR2-Mediated Reprogramming of Mitochondrial Metabolism Regulates the Sensitivity of Acute Myeloid Leukemia to Chemotherapy.

Metabolic reprogramming is central to tumorigenesis, but whether chemotherapy induces metabolic features promoting recurrence remains unknown. We established a mouse xenograft model of human acute myeloid leukemia (AML) that enabled chemotherapy-induced regressions of established disease followed by lethal regrowth of more aggressive tumor cells. Human AML cells from terminally ill mice treated with chemotherapy (chemoAML) had higher lipid content, increased lactate production and ATP levels, reduced expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), and fewer mitochondria than controls from untreated AML animals. These changes were linked to increased VEGFR2 signaling that counteracted chemotherapy-driven cell death; blocking of VEGFR2 sensitized chemoAML to chemotherapy (re-)treatment and induced a mitochondrial biogenesis program with increased mitochondrial mass and oxidative stress. Accordingly, depletion of PGC-1α in chemoAML cells abolished such induction of mitochondrial metabolism and chemosensitization in response to VEGFR2 inhibition. Collectively, this reveals a mitochondrial metabolic vulnerability with potential therapeutic applications against chemotherapy-resistant AML.Significance: These findings reveal a mitochondrial metabolic vulnerability that might be exploited to kill chemotherapy-resistant acute myeloid leukemia cells. Cancer Res; 78(3); 731-41. ©2017 AACR.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL -rearranged AML

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.