Acute Myeloid Leukemia Xenograft Mouse Research Articles

Pre-Clinical Activity of Amino-Alcohol Dimeric Naphthoquinones as Potential Therapeutics for Acute Myeloid Leukemia.

The clinical outcomes of patients with Acute Myeloid Leukemia (AML) remain unsatisfactory. Therefore the development of more efficacious and better-tolerated therapy for AML is critical. We have previously reported anti-leukemic activity of synthetic halohydroxyl dimeric naphthoquinones (BiQ) and aziridinyl BiQ. This study aimed to improve the potency and bioavailability of BiQ compounds and investigate antileukemic activity of the lead compound in vitro and a human AML xenograft mouse model. We designed, synthesized, and performed structure-activity relationships of several rationally designed BiQ analogues with amino alcohol functional groups on the naphthoquinone core rings. The compounds were screened for anti-leukemic activity and the mechanism as well as in vivo tolerability and efficacy of our lead compound was investigated. We report that a dimeric naphthoquinone (designated BaltBiQ) demonstrated potent nanomolar anti-leukemic activity in AML cell lines. BaltBiQ treatment resulted in the generation of reactive oxygen species, induction of DNA damage, and inhibition of indoleamine dioxygenase 1. Although BaltBiQ was tolerated well in vivo, it did not significantly improve survival as a single agent, but in combination with the specific Bcl-2 inhibitor, Venetoclax, tumor growth was significantly inhibited compared to untreated mice. We synthesized a novel amino alcohol dimeric naphthoquinone, investigated its main mechanisms of action, reported its in vitro anti-AML cytotoxic activity, and showed its in vivo promising activity combined with a clinically available Bcl-2 inhibitor in a patient-derived xenograft model of AML.

OTS167 blocks FLT3 translation and synergizes with FLT3 inhibitors in FLT3 mutant acute myeloid leukemia

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

Curcumin promotes cell cycle arrest and apoptosis of acute myeloid leukemia cells by inactivating AKT.

Curcumin, a phytochemical from rhizomes of the plant Curcuma longa, has been reported to exert potential anticancer properties in various cancer types, including acute myeloid leukemia (AML). However, the underlying mechanism remains poorly understood. The present study demonstrated that curcumin had a stronger cytotoxic activity against AML cells compared with three other types of phytochemicals (epigallocatechin gallate, genistein and resveratrol). Protein phosphorylation profiling using an antibody array identified that curcumin treatment increased the phosphorylation levels of 14 proteins and decreased those of four proteins. A protein-protein interaction network was constructed using the STRING database, in which AKT was identified as a hub protein with the highest connectivity (PRAS40, 4E-BP1, P70S6K, RAF-1 and p27). Western blotting results indicated that curcumin dose-dependently suppressed the phosphorylation of AKT, PRAS40, 4E-BP1, P70S6K, RAF-1 and p27 in AML cell lines (ML-2 and OCI-AML5). It was also demonstrated that curcumin regulated the cell cycle- and apoptosis-related proteins (cyclin D1, p21, Bcl2, cleaved-caspase-3 and cleaved-PARP), leading to cell cycle arrest and apoptosis in both ML-2 and OCI-AML5 cells. These effects of curcumin were enhanced by the AKT inhibitor afuresertib but were suppressed by the AKT activator SC-79, indicating that curcumin functions via AKT. In the AML xenograft mouse model, curcumin and afuresertib synergistically suppressed the engraftment, proliferation and survival of AML cells. Collectively, the present study demonstrated that curcumin exerted anti-AML roles by inactivating AKT and these findings may aid in the treatment of AML.

Investigating the Dual Targeting of BCL-2 and CD38 in Models of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is correlated with poor prognosis and a high mortality. Current AML treatment often fails to achieve complete remission and relapse is common, highlighting the need for more targeted treatments. Overexpression of BCL2 is a hallmark of AML progression and is often associated with a poor response to cytotoxic treatment. BCL2 inhibition by the BH3 mimetic Venetoclax has been shown to be effective in promoting AML cell death and recently Venetoclax has received FDA approval for the treatment of AML. However, some patients do not respond or can develop resistance, therefore multiple studies for combinational therapies for Venetoclax have been researched. We have previously demonstrated CD38 inhibition by daratumumab treatment inhibits mitochondrial transfer from mesenchymal stromal cell (MSC) to AML blasts in the bone marrow (BM) microenvironment, which results in a reduction in AML derived oxidative phosphorylation and subsequent reduced leukemia growth and improved animal survival. We therefore investigated the consequences of inhibiting CD38 using daratumumab and BCL2 using Venetoclax on the AML survival. Primary AML blasts were isolated from patient's BM. CD38 expression and BCL2 expression was assessed by flow cytometry analysis of AML. AML had significantly higher BCL2 expression and a slight increase in CD38 expression compared to CD34+ cells. Venetoclax alone caused a significant decrease in cell viability, however daratumumab or in combination with Venetoclax had no additive effect on AML survival. Since AML is highly reliant on the BM microevironment we cultured AML on MSC with either Venetoclax alone, daratumumab alone, or Venetoclax and daratumumab for 24 hours. Cells were then stained with Annexin V-FITC/PI and analysed using flow cytometry. Cells underwent significantly more apoptosis in the combination Venetoclax and daratumumab treatment when compared to control AML cells. To determine the effect of Venetoclax and daratumumab treatment in preclinical models we used an NSG xenograft mouse model of AML, we transplanted MV411-luc or patient derived AML and treated the animals with either vehicle control (PBS) daratumumab (5mg/kg) on day 7 and 14 alone, Venetoclax (100mg/kg/day) alone, or both daratumumab and Venetoclax followed by bioluminescence imaging. In vivo, treatment with combination daratumumab and Venetoclax significantly reduced tumor burden and improved survival compared to control and either drug alone in the patient derived AML xenograft mouse model and in MV411. These data support the further clinical investigation of Venetoclax and Daratumumab combination as a therapeutic approach for the treatment AML. Disclosures Bowles: AbbVie: Research Funding; Janssen: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

Nonclinical Efficacy and Toxicity and Selection of a Safe Clinical Starting Dose for an NMT Inhibitor in Development for Hematological Malignancies

N-myristoylation is the addition of the 14-carbon fatty acid myristate to proteins. This plays a fundamental role in cell signaling: Over 500 proteins are myristoylated, including all 9 Src Family Kinases (SFK) Src, Lyn, Lck, Hck, and Fgr, as well as c-Abl, Gα subunits, caspase truncated (ct-) Bid and ct-PAK2, regulating cell growth and apoptosis. Human myristoylation is performed by two ubiquitously expressed N-myristoyltransferases NMT1 and NMT2. PCLX-001 is a new, orally bioavailable, small-molecule, dual NMT inhibitor under investigation as a novel and selective treatment for B-cell malignancies.In vitro, PCLX-001 inhibits the growth of hematological cancer cells at concentrations 10-fold lower than dasatinib and ibrutinib and prevents SFK recruitment to B cell receptor signaling complex, reducing survival signals and triggering apoptosis. PCLX-001 causes complete tumor regression in NOD/SCID mouse xenograft models of Acute Myeloid Leukemia (AML), Burkitt's Lymphoma (BL), and Diffuse Large B-cell Lymphoma (DLBCL), including drug-resistant human tumor in a PDX model. In mice, PCLX-001 produced complete remission of most xenografts at ≥35 mg/kg/day but also produced some deaths associated with bacterial infection and GI hemorrhage. In an AML xenograft mouse model, the drug exposure from oral dosing required to achieve 91% tumor inhibition was 3,423 ng*hr/mL (AUC (0-tlast)). When expressed on the basis of body surface area, the efficacious dose in mice was ≥105 mg/m2. To support clinical development, we evaluated PCLX-001 for safety in 4-week oral toxicity studies with 2-week recovery periods in rats at 50, 125, and 300 mg/kg/day and dogs at 2, 4, 8, and 12 mg/kg/day, performed to Good Laboratory Practice (GLP) standards. The highest non-severely toxic dose levels (HNSTDs) were 125 mg/kg/day for male rats, 300 mg/kg/day for female rats, and 4 mg/kg/day for dogs of both sexes. At higher dose levels, dose-limiting toxicity occurred within the first few days, was similar in both species, and was attributed to GI mucosal damage and decreased hematopoiesis, with secondary complications such as dehydration and inflammation. In dogs, systemic exposure to PCLX-001 increased more-than-proportionally with dose level after the first dose and did not change with repeated daily dosing. After the last dose at the HNSTD, Cmax averaged 651 ng/mL and 24-hour AUC averaged 3,760 h*ng/mL. In rats, systemic exposure to PCLX-001 after the first dose increased approximately proportionally with dose level and was much greater in males than females at all dose levels. With repeated daily dosing, exposure decreased dramatically in male rats but less so in female rats. As a result, exposure to PCLX-001 was similar in rats of both sexes after the last dose. After the last dose at the HNSTD in males and females, Cmax was 1650 and 8510 ng/mL, respectively, and 24-hour AUC averaged 6470 and 7590 h*ng/mL, respectively. When expressed on the basis of body surface area, the HNSTDs in male rats, female rats, and dogs were 750, 1800, and 80 mg/m2, respectively. As recommended in the ICH S9 Guidance, we chose a starting dose level of 20 mg/m2 for the first clinical trial because this was approximately 1/6th of the HNSTD in dogs. Given the dissimilarity of results between species, we compared the sequence of the NMTs in each since their inhibition was the target for PCLX-001. There was no significant difference between species in their sequence homology to humanNMT1(dog was 98%, rat 97% and mouse 99%), but dogNMT2had only 78% identity to humanNMT2while rat and mouseNMT2had 95% identity with humanNMT2. The significance of this dissimilarity is still under investigation. Disclosures Weickert: Pacylex Pharmaceuticals, Inc.:Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.Dillberger:Pacylex Pharmaceuticals, Inc.:Consultancy.Mackey:Pacylex Pharmaceuticals, Inc.:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.Berthiaume:Pacylex Pharmaceuticals, Inc.:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia

AbstractFLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor–dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

Anti-CD99 scFv-ELP nanoworms for the treatment of acute myeloid leukemia

CD99 is a transmembrane glycoprotein shown to be upregulated in various malignancies. We have previously reported CD99 to be highly upregulated and present a viable therapeutic target in acute myeloid leukemia (AML). Currently, no therapy against CD99 is under clinical investigation. As a surface molecule, CD99 can be targeted with an antibody-based approach. Here, we have developed a new modality to target CD99 by engineering a fusion protein composed of a single-chain variable fragment antibody (anti-CD99 scFv) conjugated with a high molecular weight elastin-like polypeptide (ELP), A192: α-CD99-A192. This fusion protein assembles into multi-valent nanoworm with optimal physicochemical properties and favorable pharmacokinetic parameters (half-life: 16 h). α-CD99-A192 nanoworms demonstrated excellent in vitro and in vivo anti-leukemic effects. α-CD99-A192 induced apoptotic cell death in AML cell lines and primary blasts and prolonged overall survival of AML xenograft mouse model.

Targeting ferroptosis contributes to ATPR-induced AML differentiation via ROS-autophagy-lysosomal pathway.

Ferroptosis, a newly discovered form of non-apoptotic cell death, is induced by an excessive degree of iron-dependent lipid peroxide. ATPR, a novel all-trans retinoic acid (ATRA) derivative, has been extensively developed to show superior anticancer effect than ATRA in acute myeloid leukemia (AML). However, whether ferroptosis exists during ATPR treatment of AML remains unclear. Herein, we found that ferroptosis occurred in an AML xenograft mouse model of ATPR treatment. In vitro, ATPR was verified to induce ferroptosis in a dose-dependent manner by proferroptotic protein marker, lipid peroxidation, and lipid ROS, which could be significantly reversed by ferrostatin-1. Using lysosomal inhibitor chloroquine and iron chelator desferrioxamine, we further revealed that ATPR-induced ferroptosis was regulated by autophagy via iron homeostasis, especially Nrf2. Furthermore, targeting ferroptosis contributes to ATPR-induced AML differentiation. In conclusion, these results indicated that ferroptosis play an important role in ATPR-induced differentiation, and suggested that ATPR would provide a potential therapeutic value for AML treatment.

ASLAN003, a potent dihydroorotate dehydrogenase inhibitor for differentiation of acute myeloid leukemia.

Differentiation therapies achieve remarkable success in acute promyelocytic leukemia, a subtype of acute myeloid leukemia (AML). However, excluding acute promyelocytic leukemia, clinical benefits of differentiation therapies in AML are negligible except for those targeting mutant isocitrate dehydrogenase 1/2. Dihydroorotate dehydrogenase catalyzes the fourth step of the de novo pyrimidine synthesis pathway. ASLAN003 is a highly potent dihydroorotate dehydrogenase inhibitor that induces differentiation, as well as reducing cell proliferation and viability, of AML cell lines and primary AML blasts including chemoresistant cells. Apoptotic pathways are triggered by ASLAN003, and this drug also significantly inhibits protein synthesis and activates AP-1 transcription, contributing to its capacity to promote differentiation. Finally, ASLAN003 substantially reduces leukemic burden and prolongs survival in AML xenograft mice and AML patient-derived xenograft models. Notably, the drug has no evident effect on normal hematopoietic cells and exhibits excellent safety profiles in mice, even after a prolonged period of administration. Our results, therefore, suggest that ASLAN003 is an agent targeting dihydroorotate dehydrogenase with potential for use in the treatment of AML. ASLAN003 is currently being evaluated in a phase IIa clinical trial in patients with 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.

Abstract 1293: HM43239, a novel FLT3 inhibitor in overcoming resistance for acute myeloid leukemia

Abstract Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutation is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Currently, FLT3 inhibitors have shown clinical benefits in the corresponding AML patients. Activating mutations within internal tandem duplication (ITD) and tyrosine kinase domain (TKD) point mutations of FLT3 have been reported as oncogenic driver mutations in about 30% of AML. The acquired D835Y and F691L point mutations of FLT3-TKD are associated with resistance to FLT3-targeted AML therapy. In this study, we have characterized HM43239, a novel FLT3 inhibitor, and assessed its potential as a novel therapy in overcoming resistance for AML patients. Materials and Methods: In vitro site-directed competition binding assay was performed to measure interactions between HM43239 and FLT3 mutations. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML resistance cell lines. In vivo study, HM43239 was evaluated in Ba/F3 cells expressing FLT3 ITD/F691L or FLT3 ITD/D835Y xenograft mice models. Combination studies were evaluated in Acute Myeloid Leukemia xenograft mice models. Results: HM43239 potently inhibited both FLT3 ITD/D835Y and FLT3 ITD/F691L mutations in preclinical evaluation. It showed high in vitro binding affinity to both mutations, and exhibited potent inhibitory activity in in vitro and in vivo models using Ba/F3 cells expressing FLT3 ITD/D835Y or FLT3 ITD/F691L. Moreover, HM43239 could overcome the FL-induced drug resistance with a higher cytotoxic potency in MOLM-14 cells harboring FLT3 ITD. In KG-1a cells, HM43239 potently inhibited phosphorylation of SYK, STAT3 and STAT5. In addition, it inhibited the proliferation and induced the apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells), suggesting the possibility of targeting LSC. Also, HM43239 significantly inhibited p-FLT3 and p-STAT5 under normal human plasma milieu in a dose-dependent manner in Ba/F3 and MOLM-14 cell line harboring FLT3 ITD. Furthermore, the combination treatment of HM43239 with various reagents (e.g., IAP inhibitor, chemotherapy, etc) demonstrated synergistic efficacy in mouse models, xenografted with both MV-4-11 and MOLM-13 cell lines without any significant toxicity. Conclusion: Taken together, HM43239 demonstrated the potential therapeutic efficacy for the treatment of AML patients, and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: JiSook Kim, InHwan Bae, JaeYul Choi, MinJeong Kim, JooYun Byun, MiJin Moon, EunYoung Lee, Yu-Yon Kim, Hyun Jeong Kang, Eunyoung Kim, SunYoung Jung, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. HM43239, a novel FLT3 inhibitor in overcoming resistance for acute myeloid leukemia [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 1293.

Abstract 3833: Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients. Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC [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 3833.

Combination Treatment with 5F9 and Azacitidine Enhances Phagocytic Elimination of Acute Myeloid Leukemia

Combination Treatment with 5F9 and Azacitidine Enhances Phagocytic Elimination of Acute Myeloid Leukemia

Tyrosine kinase inhibitor–induced defects in DNA repair sensitize FLT3(ITD)-positive leukemia cells to PARP1 inhibitors

AbstractMutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.

Abstract 1114: IGFBP7 reduces acute myeloid leukemia stem cell survival without affecting normal hematopoiesis

Abstract Only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. This poor prognosis is mainly caused by treatment failure due to chemotherapy resistance and relapse. Leukemic stem cells (LSCs) are thought to be the major determinants of AML relapse due their potential for self-renewal and chemotherapy resistance. It has been demonstrated that LSC frequency and expression of LSC gene expression signatures is highly predictive of therapy failure in AML patients, indicating the clinical importance of LSCs. LSCs co-reside with residual normal hematopoietic stem cells (HSCs) in the diseased bone marrow. Increasing the dose of chemotherapy might eliminate LSCs, however will inevitable result in non-specific elimination of HSCs, delaying or preventing recovery of normal hematopoiesis after treatment. To significantly improve AML patients outcome, there is an urgent need for identification of alternative therapies that specifically eliminate LSCs while sparing HSC. We generated gene expression profiles of HSCs, LSCs and leukemic progenitors all derived from the same AML bone marrow and identified insulin growth factor binding protein 7 (IGFBP7) as one of the top differentially expressed genes. We found that low IGFBP7 is a feature of LSCs, associated with reduced chemotherapy sensitivity and that IGFBP7 is frequently downregulated at relapse as compared to AML diagnosis. Together, these results suggest that there is a survival advantage of IGFBP7low AML cells during chemotherapy treatment. To test whether increasing IGFBP7 levels might be a strategy to deplete leukemic (stem) cells, we overexpressed IGFBP7 or added recombinant human IGFBP7 (rhIGFBP7) to primary AML cells. Strikingly, IGFBP7 overexpression or addition of rhIGFBP7 led to induction of differentiation and cell death in AML patient cells. Moreover, rhIGFBP7 reduces leukemic engraftment in an AML xenograft mouse model, reverses a stem-like gene signature, and inhibits AML blast and leukemic stem/progenitor cell survival in vitro and in vivo, while it had no influence on normal hematopoietic (stem) cell survival. Our findings define an association between chemotherapy sensitivity and IGFBP7 expression levels in primary AML cells, and indicate that treatment of AML patients with a combination of rhIGFBP7 and chemotherapy might reduce AML residual disease and LSC survival. Citation Format: Han J. Verhagen, Noortje van Gils, Anna van Rhenen, Arjo Rutten, Marjon Smit, Mei-Ling Tsui, Louise L. de Vos Klootwijk, Renee X. Menezes, Meyram Cil, Margaretha G. Roemer, Fabio Brocco, Fedor Denkers, Eline Vermue, Stan Heukelom, Sonja Zweegman, Jeroen J. Janssen, Gert J. Ossenkoppele, Gerrit Jan Schuurhuis, Linda Smit. IGFBP7 reduces acute myeloid leukemia stem cell survival without affecting normal hematopoiesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1114.

Cotargeting BCL-2 and PI3K Induces BAX-Dependent Mitochondrial Apoptosis in AML Cells.

Inhibitors targeting BCL-2 apoptotic proteins have significant potential for the treatment of acute myeloid leukemia (AML); however, complete responses are observed in only 20% of patients, suggesting that targeting BCL-2 alone is insufficient to yield durable responses. Here, we assessed the efficacy of coadministration of the PI3K/mTOR inhibitor GDC-0980 or the p110β-sparing PI3K inhibitor taselisib with the selective BCL-2 antagonist venetoclax in AML cells. Tetracycline-inducible downregulation of BCL-2 significantly sensitized MV4-11 and MOLM-13 AML cells to PI3K inhibition. Venetoclax/GDC-0980 coadministration induced rapid and pronounced BAX mitochondrial translocation, cytochrome c release, and apoptosis in various AML cell lines in association with AKT/mTOR inactivation and MCL-1 downregulation; ectopic expression of MCL-1 significantly protected cells from this regimen. Combined treatment was also effective against primary AML blasts from 17 patients, including those bearing various genetic abnormalities. Venetoclax/GDC-0980 markedly induced apoptosis in primitive CD34+/38-/123+ AML cell populations but not in normal hematopoietic progenitor CD34+ cells. The regimen was also active against AML cells displaying intrinsic or acquired venetoclax resistance or tumor microenvironment-associated resistance. Either combinatorial treatment markedly reduced AML growth and prolonged survival in a systemic AML xenograft mouse model and diminished AML growth in two patient-derived xenograft models. Venetoclax/GDC-0980 activity was partially diminished in BAK-/- cells and failed to induce apoptosis in BAX-/- and BAX-/-BAK-/- cells, whereas BIM-/- cells were fully sensitive. Similar results were observed with venetoclax alone in in vitro and in vivo systemic xenograft models. Collectively, these studies demonstrate that venetoclax/GDC-0980 exhibits potent anti-AML activity primarily through BAX and, to a lesser extent, BAK. These findings argue that dual BCL-2 and PI3K inhibition warrants further evaluation in AML.Significance: Combined treatment with clinically relevant PI3K and BCL-2 inhibitors may prove effective in the treatment of acute myeloid leukemia. Cancer Res; 78(11); 3075-86. ©2018 AACR.

Mebendazole exhibits potent anti-leukemia activity on acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most common types of acute leukemia in adults with the lowest survival rate of all leukemia. Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure. Thus, finding new drugs with anti-leukemia activities and minimal side effect is urgently needed. Here through screening more than 1000 drugs approved by the Food and Drug Administration (FDA) of United States, the anthelmintic drug mebendazole (MBZ) was found to inhibit the growth of AML cell lines (THP-1, U937, NB4 and K562) and bone marrow mononuclear cells (BM-MNCs) from AML patients at pharmacologically achievable concentrations. In contrast, similar concentration of MBZ had little inhibitory effect on the growth of normal peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC). In addition, MBZ induced mitotic arrest and mitotic catastrophe in AML cells based on nuclear morphology, cell cycle distribution, mitotic marker analyses and the number of multinucleated cells and apoptotic cells. Furthermore, MBZ treatment inhibited activation of Akt and Erk in AML leukemic cells. Finally, MBZ repressed the progression of leukemic cells in vivo and prolonged survival in AML xenograft mouse model. Taken together, our results suggest that MBZ could be a potential new therapeutic agent for the treatment of AML patients.

Targeting FLT3 in acute myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells

BackgroundChimeric antigen receptor-engineered T (CAR-T) cells have extraordinary effect in treating lymphoblastic leukemia. However, treatment of acute myeloid leukemia (AML) using CAR-T cells remains limited to date. Leukemogenesis always relates with the abnormalities of cytogenetics, and nearly one third of AML patients have activating mutations in Fms-like tyrosine kinase 3 (FLT3) which reminded poor prognosis. Considering the FLT3 expressed in AML patients’ blast cells, it may be a new candidate target for CAR-T therapy to treat FLT3+ AML, especially patients harboring FLT3-ITD mutation.MethodsThe FLT3L CAR-T using FLT3 ligand as recognizing domain was constructed. The specific cytotoxicity against FLT3+ leukemia cell lines, primary AML cells, and normal hematopoietic progenitor stem cells (HPSCs) in vitro were evaluated. In addition, FLT3+ AML mouse model was used to assess the effect of FLT3L CAR-T therapy in vivo.ResultsFLT3L CAR-T cells could specifically kill FLT3+ leukemia cell lines and AML patients’ bone marrow mononuclear cells in vitro (with or without FLT3 mutation) and have more potent cytotoxicity to FLT3-ITD cells. In a human FLT3+ AML xenograft mouse model, FLT3L CAR-T cells could significantly prolong the survival of mice. Furthermore, it was found that FLT3L CAR-T cells could activate the FLT3/ERK signaling pathway of FLT3+ leukemia cells with wild-type FLT3; meanwhile, it had no inhibitory effects on the colony formation of CD34+ stem cells derived from normal human umbilical cord blood.ConclusionsThe ligand-based FLT3L CAR-T cells could be a promising strategy for FLT3+ AML treatment, especially those carried FLT3 mutation.

Regulation of human stem and progenitor cells by acute myeloid leukaemia in the human niche

In acute myeloid leukemia (AML) the suppression of normal blood production is a major factor contributing to treatment complications and mortality rates. Recently it was demonstrated that cell cycle inhibition of haematopoietic stem and progenitor cells (HSPCs) may be the underlying cause of suppression in both AML xenograft and transgenic mouse models. However, the mechanism in human HSPCs is still poorly understood. Using a fully humanized ex vivo system comprising AML cell lines, umbilical cord blood derived CD34+ HSPCs and bone marrow mesenchymal stroma cells (MSCs), we studied the interactions between AML, normal HSPCs and their stromal niche. In our ex vivo system, AML but not normal white blood cells rapidly suppress the proliferation and differentiation of normal HSPCs via cell cycle inhibition of both (CD34+CD38+) progenitors and (CD34+CD38-CD45RA-CD90+) stem cells. In line with the finding of the in vivo AML models, AML did not induce apoptosis in HSPCs. Transcriptomic interrogation of a stroma cell line after AML exposure identified a hypoxia-response signature and an upregulation of Hypoxia inducible factor -1 alpha (HIF-1α) target genes. Hypoxia-induced stabilization of HIF-1α in MSCs recapitulated AML induced HSPC suppression, which was reversible by HIF-1α knockdown. Furthermore, we identified Stanniocalcin 1 (STC1), a small secreted glycoprotein, to be upregulated via HIF-1α dependent mechanisms in MSCs after AML exposure. Recombinant STC1 alone reduced HSPC proliferation and differentiation, using a STC1 neutralizing antibody, we were able to significantly reduce the AML-induced cell cycle inhibition of stem cells and increase the number of progenitors. In summary, we developed a fully humanized ex vivo system to study the interaction between AML, normal HSPCs and the stroma niche. We identified HIF-1α stabilization and its downstream target STC1 in MSCs as key contributors to the suppression of HSPC differentiation and proliferation, providing a potential route to release normal blood production from AML-induced suppression.

JAK1/2 and BCL2 inhibitors synergize to counteract bone marrow stromal cell–induced protection of AML

AbstractThe bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.