Against Acute Myeloid Leukemia Patients Research Articles

Repurposing Atovaquone as a Therapeutic against Acute Myeloid Leukemia (AML): Combination with Conventional Chemotherapy Is Feasible and Well Tolerated

Survival of pediatric AML remains poor despite maximized myelosuppressive therapy. The pneumocystis jiroveci pneumonia (PJP)-treating medication atovaquone (AQ) suppresses oxidative phosphorylation (OXPHOS) and reduces AML burden in patient-derived xenograft (PDX) mouse models, making it an ideal concomitant AML therapy. Poor palatability and limited product formulations have historically limited routine use of AQ in pediatric AML patients. Patients with de novo AML were enrolled at two hospitals. Daily AQ at established PJP dosing was combined with standard AML therapy, based on the Medical Research Council backbone. AQ compliance, adverse events (AEs), ease of administration score (scale: 1 (very difficult)-5 (very easy)) and blood/marrow pharmacokinetics (PK) were collected during Induction 1. Correlative studies assessed AQ-induced apoptosis and effects on OXPHOS. PDX models were treated with AQ. A total of 26 patients enrolled (ages 7.2 months-19.7 years, median 12 years); 24 were evaluable. A total of 14 (58%) and 19 (79%) evaluable patients achieved plasma concentrations above the known anti-leukemia concentration (>10 µM) by day 11 and at the end of Induction, respectively. Seven (29%) patients achieved adequate concentrations for PJP prophylaxis (>40 µM). Mean ease of administration score was 3.8. Correlative studies with AQ in patient samples demonstrated robust apoptosis, OXPHOS suppression, and prolonged survival in PDX models. Combining AQ with chemotherapy for AML appears feasible and safe in pediatric patients during Induction 1 and shows single-agent anti-leukemic effects in PDX models. AQ appears to be an ideal concomitant AML therapeutic but may require intra-patient dose adjustment to achieve concentrations sufficient for PJP prophylaxis.

Vitamin C promotes anti-leukemia of DZNep in acute myeloid leukemia

The epigenetic treatment by 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, shows great potential against acute myeloid leukemia (AML). However, the variant sensitivity and incomplete response to DZNep are commonly observed. Here, we reveal that vitamin C (Vc) dramatically promotes DZNep response against leukemic cells in different cell lines and primary AML samples. Vc enhances apoptosis and differentiation induced by DZNep in different AML cell lines in vitro and reduces leukemia progression in vivo. At the molecular level, Vc downregulates an enzyme of serine synthesis named D-3-phosphoglycerate dehydrogenase (PHGDH), as well as BCL2, an anti-apoptotic gene. Over-expression of PHGDH reverses the Vc-enhanced anti-leukemic effect of DZNep in AML cells. Therefore, our findings provide an effective approach to reduce the resistance against epigenetic treatment by Vc, which shows a potential improvement of their combination in AML patients.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / − 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5′ primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

Melanoma cells can be eliminated by sialylated CD43 × CD3 bispecific T cell engager formats in vitro and in vivo.

Targeted cancer therapy with monoclonal antibodies has proven successful for different cancer types but is limited by the availability of suitable antibody targets. CD43s, a unique sialylated form of CD43 expressed by hematologic malignancies, is a recently identified target and antibodies interacting with CD43s may have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was derived from a high-risk AML patient who successfully cleared leukemia after allogeneic stem cell transplantation. Here we observed that AT1413 binds also to certain non-hematopoietic tumor cells, particularly melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody-dependent cellular cytotoxicity against short-term cultured patient-derived melanoma samples. However, AT1413 was unable to affect the growth of melanoma cells in vivo. To increase the efficacy of AT1413 as a therapeutic antibody, we generated two different formats of bispecific T-cell engaging antibodies (TCEs): one binding bivalently (bTCE) and the other monovalently (knob-in-hole; KiH) to both CD43s and CD3ε. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with differences in potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth in these mice. These data indicate a broad therapeutic potential of AT1413 that includes AML and CD43s-expressing solid tumors that originate from CD43-negative tissues.

Clonal Expansion of Mutant p53 Clones By MDM2 Inhibition in Acute Myeloid Leukemias

MDM2 inhibition by small molecules as a means of restoring p53 function has shown clinical activity against acute myeloid leukemia (AML) (Andreeff, Clin Cancer Res 2015). However, we and others have found increased variant allele frequencies (VAFs) of TP53 mutations in AML cells after treatment with MDM2 inhibitors, either as monotherapy or in combination with other agents (Daver ASH 2019), which suggests that MDM2 inhibition selects preexisting clones or generates de novo clones with TP53 mutations. We performed a long-term culture of AML cells (MOLM-13, an AML cell line with wild-type p53 and FLT3-ITD) treated with increasing concentrations of an MDM2 inhibitor idasanutlin (up to 320 nM, less than 10% concentration of Cmax) (Selleck). We obtained MDM2 inhibitor-resistant (R) AML cells after 96 days of the drug exposure and found that the resistant cells harbor hotspot TP53 p.R248W (R248W) mutation. We next isolated single cell clones from MOLM-13 R cells by limiting dilution, and obtained twelve subclones (subclones #1-12 in order of development). All clones carried the same R248W mutation. To determine clonal patterns of these cells, we performed single cell DNA sequencing (scDNAseq) of MOLM-13 parental, R and subclone #1 and #2 (SC1 and SC2) cells using the MissionBio Tapestry system covering 125 amplicons of 19 genes frequently mutated in AML. scDNAseq identified FLT3-ITD mutations in all cells analyzed, as expected. In the parental cells we identified only 0.02% cells (1/ 5,240) with the identical R248W mutation found in MOLM-13 R cells. MOLM-13 R cells had only 0.6% of wild-type TP53 cells, 51% carrying R248W only, and 43% R248W/R213* mutation (R248W/R213*). SC1 and SC2 cells had 1% and 99% of R248W and R248W/R213* clones, respectively (Fig.1). Seven other mutations were detected by scDNAseq. Results suggest that MDM2 inhibition can accelerate the selection of TP53-mutant AML cells in vitro. Of note, the parental cells had remained mostly p53 wild-type, where the subclone with mutant R248W did not have a growth advantage over other cells. Next we analyzed patient samples enrolled in the phase 1 clinical trial (NCT02319369) for the MDM2 inhibitor milademetan (DS-3032b; Daiichi-Sankyo) in relapsed/refractory AML or high-risk MDS patients. Fifty seven patients were treated with single agent milademetan in the study. All but one patient had wild-type TP53 as determined by NGS at baseline. One patient (1.8%) had a TP53 p.R213* mutation at baseline with VAF of 91%. Four patients (7%) developed different TP53 mutations (R248Q, R248W, P250fs, V122fs and V274L), with increasing VAFs that were not detected at baseline to 19% average, ranging from 11% to 28% post treatment, One patient (anonymized ID 1001-1005) developed both, R248Q and R248W mutations, detected at cycle 2 day1 (C2D1, day 29). The pre-existing R213* mutation detected in one patient persisted with increased VAF after treatment (91% to 100%). To detect p53 mutations with higher sensitivity than NGS, we performed droplet digital PCR (ddPCR) for R248W/Q and R273H in samples from two patients. ddPCR detected 0.46% and 0.62% of R248Q and R248W mutations, respectively, at baseline, which were not detected by NGS, with increased VAFs of 18.2 and 27.6% at C2D1, respectively. ddPCR detected additional R273H mutations with VAFs of 0.08% and 0.18% at baseline, and 2.2% and 2.6% in these patients at C2D1, respectively. Conclusion: Data suggest that MDM2 inhibition selects rare AML subpopulations with TP53 mutations and careful monitoring of patients treated with MDM2 inhibitors with sensitive methods to detect TP53 mutant clones is warranted. This finding also points to the need to develop strategies to prevent/suppress these clones early on. Disclosures Kumar: Daiichi-Sankyo Inc.: Current Employment. Patel:Daiichi-Sankyo Inc.: Current Employment. Dos Santos:Daiichi Sankyo, Inc.: Current Employment. DiNardo:Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; ImmuneOnc: Honoraria; Calithera: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Syros: Honoraria. Andreeff: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 & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding.

Abstract 542: T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo

Abstract Introduction CD43s, a unique sialylated form of CD43 expressed by myeloid malignancies, is a novel target and AT1413, an antibody targeting CD43s was shown to have therapeutic potential against acute myeloid leukemia (AML) and myelodysplastic syndrome. CD43s is recognized by the human antibody AT1413, that was isolated from a high-risk AML patient who successfully cleared the leukemia after allogeneic stem cell transplantation. Because CD43 is also expressed in non-hematopoietic cells we studied whether CD43s is also present and can also be targeted on non-hematopoietic tumors. Materials and methods AT1413 binding on a panel of tumor cell lines was analyzed by flow cytometry. AT1413 was constructed into a bispecific T-cell engaging format (AT1413 bTCE) by linking the full-length AT1413 IgG to two single chain variable fragments against CD3ϵ with a combination of site-specific enzymatic and chemical coupling. A monovalent T-cell engager was produced as heterotrimer consisting of one AT1413 monovalent chain and one anti-CD3 scFV fused to AT1413 monovalent chain. Point mutations in the IgG heavy chain were introduced to prevent interactions between AT1413 T-cell engagers and Fc-receptors. The cytotoxicity-inducing activities were established using PBMCs as effector and tumor cells as target cells using standard cytotoxic assays in vitro and in vivo in a mouse model carrying human immune cells. Results We observed that AT1413 binds to non-hematopoietic tumor cells, such as melanoma and breast cancer. AT1413 immune precipitated CD43s from melanoma cells confirming that it recognizes the same target on melanoma as on AML. AT1413 induced antibody dependent cellular cytotoxicity against melanoma cell lines and primary melanoma samples. However, AT1413 was unable to affect growth of melanoma cells in vivo. To increase the efficacy of AT1413 it was formatted as a bispecific T-cell engaging antibody (TCE): one binding bivalently (bTCE) to CD43s and monovalently to CD3ϵ and the other monovalently (KiH) to both CD43s and CD3ϵ. In vitro, these TCEs redirected T-cell cytotoxicity against melanoma cells with different potencies. To investigate their effects in vivo, we grafted mice that harbor a human immune system with the melanoma cell line A375. Treatment with both AT1413 bTCE and AT1413 KiH significantly reduced tumor outgrowth. Conclusion AT1413 recognizes a sialylated epitope on CD43 shared by melanoma, AML and MDS cells. Two different bispecific TCE forms of AT1413 induce strong anti-tumor cytotoxic activities in vitro and in vivo. These data indicate a broad therapeutic potential of AT1413. Citation Format: Greta de Jong, Lina Bartels, Martijn Kedde, Els Verdegaal, Marijn A. Gillissen, Sophie E. Levie, Madalina G. Cercel, Susan E. van Hal-van Veen, Christien Fatmawati, Dorien van de Berg, Etsuko Yasuda, Yvonne Claassen, Arjen Q. Bakker, Remko Schotte, Julien Villaudy, Koen Wagner, Hergen Spits, Mette D. Hazenberg, Pauline M. van Helden. T-cell engager bispecific formats of an AML patient-derived antibody targeting a unique sialylated CD43 epitope induce kill of melanoma cells in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 542.

Alpha-Particle Therapy for Acute Myeloid Leukemia

Early studies using the humanized anti-CD33 monoclonal antibody lintuzumab labeled with β-emitters showed significant activity against acute myeloid leukemia (AML) but produced prolonged myelosuppression necessitating hematopoietic cell transplantation (HCT). Targeted α-particle therapy may produce more efficient tumor killing while sparing normal cells. An initial phase I trial of bismuth-213 (213Bi)-lintuzumab in relapsed and refractory (R/R) AML provided proof-of-principle for systemically administered α-particle therapy (Jurcic, Blood 2002). 213Bi-lintuzumab demonstrated rapid targeting of disease sites without significant extramedullary toxicity. Target-to-whole body dose ratios were greatly enhanced compared to β-emitting immunoconjugates. Anti-leukemic effects were seen across all dose levels with a decrease in marrow blasts in 78% of patients. In a subsequent phase I/II trial, 213Bilintuzumab was administered following partial cytoreduction with cytarabine (Rosenblat, Clin Cancer Res 2010). Among patients receiving the maximum tolerated dose of 37 MBq/kg or higher, responses were seen in 24% of patients. The 46-minute half-life of 213Bi, however, remained an obstacle to its widespread use. Therefore, a more potent second-generation construct containing actinium-225 (225Ac) (t½ = 10 days), which generates four α-particle emissions, was developed. A phase I study demonstrated that a single dose of 225Aclintuzuamb was safe at doses of 111 kBq/kg or less and produced marrow blast reductions in 67% of evaluable patients with R/R AML (Jurcic, ASH 2011). Dose-limiting toxicity was prolonged myelosuppression, and no evidence of radiation-induced nephrotoxicity was seen. Based on these findings, 225Ac-lintuzumab was investigated in a multicenter phase I/II trial in combination with low-dose cytarabine for older patients with untreated AML (Jurcic, SNMMI 2017). During the first cycle of therapy, two fractions of 225Ac-lintuzumab (18.5-74 kBq/kg/fraction) were administered one week apart after completion of chemotherapy. Five of 18 patients (28%) had objective responses. All responses occurred after the first cycle. The baseline peripheral blood blast count was a strong predictor of outcome, as responses were seen only in patients with peripheral blast counts < 200/μL. This is most likely explained by preferential antibody binding to peripheral sites in patients with higher circulating blast counts, leading to decreased marrow targeting. Because of this observation, a multicenter phase II trial of 225Ac-lintuzumab monotherapy using hydroxyurea to lower peripheral blast counts if needed was undertaken in older AML patients (Finn, ASH 2017). Objective responses were seen in nine of 13 patients (69%) after two doses of 225Ac-lintuzumab (74 kBq/kg) administered one week apart. Myelosuppression, however, was considered to be longer than acceptable in this population, and additional patients were treated with two fractions of 55.5 kBq/kg each. 225Ac-lintuzumab has shown significant activity in AML both alone and in combination with chemotherapy. Future development of 225Ac-lintuzumab includes combinations with standard chemotherapy and novel targeted agents for R/R AML, treatment for measurable residual disease in AML, and conditioning before HCT in patients with high-risk myelodysplastic syndromes.

Off-Label Use of Venetoclax Combination Therapy in Relapsed/Refractory Acute Myeloid Leukemia: A Single Institution Experience

Off-Label Use of Venetoclax Combination Therapy in Relapsed/Refractory Acute Myeloid Leukemia: A Single Institution Experience

Abstract 1323: CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients

Abstract CG-806 is a pan-FLT3 / pan-BTK inhibitor that is more potent (IC50 = 0.08 µM, n=265, p &amp;lt; 0.001) than other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib, crenolanib and gilteritinib against acute myeloid leukemia (AML) primary patient samples containing wild-type or mutated FLT3. CG-806 has significant activity against AML cell lines with or without FLT3 internal tandem duplication (ITD) / tyrosine kinase domain (TKD) mutations and in mouse AML xenograft models. Oral CG-806 has a desirable safety profile in the pre-IND studies of rodent and dog 28-day GLP toxicology, rodent respiratory and central nervous system safety, and the bacterial reverse mutation assay. The current study explored the relationship between genetic abnormalities in bone marrow and peripheral mononuclear cells isolated from AML patients and sensitivity to CG-806 using an ex vivo cytotoxicity assay. To correlate CG-806 sensitivity with clinical status, gene abnormalities and expression levels, whole exome sequencing (n=118) and RNA sequencing (n=111) were performed. CG-806 was equally potent against cells from patients in the adverse, intermediate and favorable risk groups (2017 ELN risk stratification), and cells from patients with relapsed or transformed AML (WHO classification) were as sensitive as those from patients with de novo AML. CG-806 had equivalent potency in cases of TP53 WT and TP53 mutations, whereas cases with TP53 mutations were resistant (FDR-corrected p&amp;lt;0.1) to most other FLT3 inhibitors including midostaurin, sorafenib, sunitinib, dovitinib, quizartinib and crenolanib. CG-806 had similar potency in cases with ASXL1 or SRSF2 mutations compared to WT, whereas sunitinib and crenolanib appeared resistant to ASXL1 and SRSF2 mutations, respectively. As expected, patient samples with FLT3-ITD mutation were more sensitive to CG-806 as compared to FLT3 WT (FDR-corrected p&amp;lt;0.01); in addition, cases with high ITD allelic ratio, including concurrent mutations with NPM1 and DNMT3A, had greater sensitivity than cases with low allelic ratio. Most unexpectedly, all 6 specimens containing IDH1 R132 mutations demonstrated significantly greater sensitivity to CG-806 relative to WT (FDR-corrected p&amp;lt;0.01), yet there was no increased sensitivity of IDH-2 mutant cells to CG-806. In conclusion, CG-806 demonstrated potency in primary AML patient samples across all AML subgroups including relapsed/refractory/transformed AML and those with genetic abnormalities related to poor prognosis. While patient samples with FLT3-ITD mutations were expected to have greater sensitivity to CG-806, the most surprising correlation was the sensitivity of patient samples with IDH1 R132 mutations. These features of CG-806 warrant investigation in the clinical setting. Citation Format: Hongying Zhang, Andrea Local, Jeffrey W. Tyner, Stephen E. Kurtz, Beth Wilmot, Shannon Mcweeney, Brian J. Druker, Stephen B. Howell, William G. Rice. CG-806, a pan-FLT3 / pan-BTK inhibitor, demonstrates superior potency against cells from IDH-1 mutant and other non-favorable risk groups of AML patients [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 1323.

PS984 CANNABINOIDS EXERT A POTENT ANTI‐LEUKEMIC EFFECT AFFECTING SIGNALING PATHWAYS INVOLVED IN TUMOR CELL METABOLISM

Background:Cannabinoids exert their activity upon interaction with cannabinoid receptors: CB1, mostly expressed in the central nervous system and CB2, mainly found in hematopoietic cells. We have described the effect of cannabinoid derivatives in multiple myeloma (Barbado et al, 2018). In this work, we present evidence that cannabinoids acts as a cytotoxic agent against acute myeloid leukemia (AML) blasts without affecting normal hematopoietic cells.Aims:Our main goal is to determine the mechanism of action by which cannabinoids exert their antileukemic effect.Methods:Cell viability was determined by MTT assay and flow cytometry analysis of Annexin V/7‐AAD using 6 AML cell lines, primary hematopoietic stem cells (HSC), lymphocytes from healthy donors and 23 AML patients. mRNA expression profile of HL60 AML cells treated with WIN‐55 vs untreated cells was studied by Clariom S Assays Human (Affimetrix) and Transcriptome Analysis Console (TAC) Software (Thermo Fisher). Western blot and immunoflurescence assays were performed to determine protein expression upon WIN‐55 exposure, including endoplasmic reticulum stress protein. TMRE and MitoSOXTM Red was used to determine mitochondrial damage. The contribution of compounds (glycine, serine, N‐Acetyl‐cysteine, 2‐mercaptoethanol, oxaloacetic acid and methyl pyruvate) involved in key metabolic processes affected upon WIN‐55 exposure were also assessed. Glycolytic flux was assessed by ECAR in a XF24 flux analyzer (Seahorse Biosciences). NAD/NADH and ADP/ATP ratio and Hexokinase activity was also measured. Ceramide and other sphingolipids were quantitated by HPLC‐MS/MS and immunohistochemistry. HL60‐xenotransplant NSG mouse model were established, bone marrow (BM) infiltration confirmed by BM aspirate and subsequent flow cytometry, and WIN‐55 or citarabine treatments was administered. The effect of WIN‐55 on normal hematopoiesis of healthy BALB‐C mice was assessed by flow cytometry and hemogram.Results:We confirmed a potent cytotoxic effect of WIN‐55 on AML cell lines while normal HSC remained unaffected. mRNA expression profile using showed that the pathways mainly affected upon WIN‐55 exposure were unfolded protein response (UPR) and XBP1 activates chaperon genes, PI3K‐Akt‐mTOR, VEGFA‐VEGFR2 and Adipogenesis Signaling Pathway. Using WB assays we confirmed that WIN‐55 triggered caspases activation and we detected ceramide accumulation in AML cells. Also studies on p‐PERK, p‐IRE1 and CHOP confirmed an increased endoplasmic reticulum stress upon exposure to cannabinoids. Pathways involved in cell proliferation/survival including p‐JNK, p‐Erk1/2, p‐p38‐MAPK and p‐AKT were affected. An early mitochondrial damage was also observed in leukemic cells upon exposure to the drug. Ceramide synthesis inhibition prevented less than 10% of the WIN‐55 induced‐apoptosis. By contrast, the proapoptotic effect of cannabinoids on AML cells was abolished upon co‐culture with either CB2 receptor antagonists, pancaspase inhibitors, olaparib (a PARP inhibitor) or glycine. Cannabinoids did not affect the viability of HSC from healthy donors neither in vitro not in vivo, resulting in a lack of hematopoietic toxicity in healthy treated mice. Finally, WIN‐55 significantly prolonged the survival of AML xenografted mice.Summary/Conclusion:Cannabinoids have a potent and selective anti‐leukemic effect affecting several signaling and metabolic pathways. The role of PARP is essential in this apoptotic effect, which is mediated by CB2 receptor. Differences in the metabolic requirements between normal and leukemic cells account for this differential effect.

Alpha-Particle Therapy for Acute Myeloid Leukemia

Early studies using the humanized anti-CD33 monoclonal antibody lintuzumab labeled with β-emitters showed significant activity against acute myeloid leukemia (AML) but produced prolonged myelosuppression necessitating hematopoietic cell transplantation (HCT). Targeted α-particle therapy may produce more efficient tumor killing while sparing normal cells. An initial phase I trial of bismuth-213 (213Bi)-lintuzumab in relapsed and refractory (R/R) AML provided proof-of-principle for systemically administered α-particle therapy (Jurcic, Blood 2002). 213Bi-lintuzumab demonstrated rapid targeting of disease sites without significant extramedullary toxicity. Target-to-whole body dose ratios were greatly enhanced compared to β-emitting immunoconjugates. Anti-leukemic effects were seen across all dose levels with a decrease in marrow blasts in 78% of patients. In a subsequent phase I/II trial, 213Bilintuzumab was administered following partial cytoreduction with cytarabine (Rosenblat, Clin Cancer Res 2010). Among patients receiving the maximum tolerated dose of 37 MBq/kg or higher, responses were seen in 24% of patients. The 46-minute half-life of 213Bi, however, remained an obstacle to its widespread use. Therefore, a more potent second-generation construct containing actinium-225 (225Ac) (t½ = 10 days), which generates four α-particle emissions, was developed. A phase I study demonstrated that a single dose of 225Aclintuzuamb was safe at doses of 111 kBq/kg or less and produced marrow blast reductions in 67% of evaluable patients with R/R AML (Jurcic, ASH 2011). Dose-limiting toxicity was prolonged myelosuppression, and no evidence of radiation-induced nephrotoxicity was seen. Based on these findings, 225Ac-lintuzumab was investigated in a multicenter phase I/II trial in combination with low-dose cytarabine for older patients with untreated AML (Jurcic, SNMMI 2017). During the first cycle of therapy, two fractions of 225Ac-lintuzumab (18.5-74 kBq/kg/fraction) were administered one week apart after completion of chemotherapy. Five of 18 patients (28%) had objective responses. All responses occurred after the first cycle. The baseline peripheral blood blast count was a strong predictor of outcome, as responses were seen only in patients with peripheral blast counts < 200/μL. This is most likely explained by preferential antibody binding to peripheral sites in patients with higher circulating blast counts, leading to decreased marrow targeting. Because of this observation, a multicenter phase II trial of 225Ac-lintuzumab monotherapy using hydroxyurea to lower peripheral blast counts if needed was undertaken in older AML patients (Finn, ASH 2017). Objective responses were seen in nine of 13 patients (69%) after two doses of 225Ac-lintuzumab (74 kBq/kg) administered one week apart. Myelosuppression, however, was considered to be longer than acceptable in this population, and additional patients were treated with two fractions of 55.5 kBq/kg each. 225Ac-lintuzumab has shown significant activity in AML both alone and in combination with chemotherapy. Future development of 225Ac-lintuzumab includes combinations with standard chemotherapy and novel targeted agents for R/R AML, treatment for measurable residual disease in AML, and conditioning before HCT in patients with high-risk myelodysplastic syndromes.

Immunocytokines with Target Cell-Restricted IL-15 Activity for Induction of NK Cell Reactivity Against Acute Myeloid Leukemia (AML)

Introduction: The efficacy of monoclonal antibodies (mAbs), which have substantially improved treatment options for cancer patients, largely relies on their ability to induce antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Recently, we have introduced Fc-optimized (SDIE modification) antibodies targeting CD133 and CD135 (FLT3) with improved capacity to induce NK cell reactivity against AML cells (Koerner et al, Leukemia 2017; Hofmann et al, Leukemia 2012). Our FLT3 mAb termed FLYSYN is currently clinically evaluated in AML patients with minimal residual disease (NCT02789254). Notably, NK cell reactivity can be substantially increased by the cytokine IL-15, but clinical application of truly effective doses is currently prevented by substantial side effects due to unspecific immune activation (Conlon et al, JCO 2015). To overcome this limitation and to strengthen therapeutic efficacy, we fused our Fc-optimized CD133 and CD135 mAbs to an IL-15 mutant with abolished binding to IL-15 receptor α (IL-15Rα). The resulting modified immunocytokines (MIC) should substitute trans-presentation of IL-15 by binding to their target antigens on leukemic cells which facilitates stimulation of IL-15Rβ/γ on NK cells.Methods: Comparative analysis of MIC133/MIC135 binding to target cells, target antigen expression and induction of antigen shift was performed by flow cytometry using primary AML cells and target antigen transfected cell lines. NK cell activation was monitored by flow cytometric analyses of activation markers such as CD69 and CD25. Cytokine release, in particular that of IFN-γ, was measured by ELISA. Target cell killing in cocultures of healthy peripheral blood mononuclear cells (PBMC) with primary AML cells or target antigen transfected cell lines was studied by Europium, Xcelligence and flow cytometry based assays. Toxicity against healthy FLT3 expressing cells was studied by flow cytometric analysis of monocytes, dendritic cells and CD34+ cells within healthy PBMC or bone marrow. For in vivo analysis, MIC135 was tested in a NOD.Cg-Prkdc(scid)IL2rg(tmWjl)/Sz xenotransplantation model by inducing leukemia with primary AML cells and polyclonal NK cells as effector cells.Results: Functional analyses confirmed target antigen-restricted binding of MIC133/MIC135 with saturating doses reached at approximately 1µg/ml. FLT3 was found to be expressed on primary AML cells with significantly higher extent and to be less susceptible to antigen shift compared to CD133. Analysis of activation and cytokine release, the latter being particularly relevant for side effects, demonstrated that MIC proteins stimulate NK cells in a target cell-restricted manner and to a profoundly greater extent than their Fc-optimized counterparts without IL-15. In line, target cell killing induced by either MIC was clearly superior to that of the respective Fc-optimized CD133 and FLT3 mAbs as revealed by various experimental systems using primary AML cells. MIC135, which was chosen for further development due to its superior characteristics described above, did not induce unwanted effects against healthy FLT3 expressing cells and potently reduced leukemic burden in a NSG xenotransplantation model with primary AML and polyclonal NK cells.Conclusion: In summary, MIC stimulate NK cells in a target cell-restricted manner, clearly outperform Fc-optimized antibodies and thus constitute a promising treatment option for AML. DisclosuresJung:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Salih:Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Inecalcitol Enhances Daratumumab-Induced Antibody-Dependent Cell Cytotoxicity Towards Multiple Myeloma and Acute Myeloid Leukemia Cell Lines

Daratumumab (DARA), the first monoclonal antibody directed against CD38, received accelerated approval in 2015 and regular approval in 2016 by the Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma (MM) (Bhatnagar et al., 2017); its therapeutic indications were recently extended to first-line newly diagnosed MM patients in combination with standards of care following a positive clinical trial in this setting (Mateos et al., 2018). Therefore, DARA has brought extensive evidence that targeting CD38 is a highly successful strategy against MM (Frerichs et al., 2018; Plesner & Krejcik, 2018; Touzeau & Moreau, 2017). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016); furthermore, low level of CD38 expression has recently been identified as an adverse prognostic factor with high significance (p < 0.001) among MM patients (Arana et al., 2018). Besides MM, there are growing preclinical hints that DARA can be efficacious against acute myeloid leukemia (AML) cell lines (Fatehchand et al., 2016; Buteyn et al., 2018) or even T-cell acute lymphoblastic leukemia cells obtained from children patients (Bride et al., 2018), provided that enough CD38 molecules are present at the surface of the leukemic cells.Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating properties and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), has been shown to stimulate CD38 expression levels in 37 out of 38 MM cell lines (ASH 2017) and in 10 out of 11 AML cell lines (ASH 2016, AACR 2018). The next question was to know if this increase in CD38 expression induced by INE resulted in an increase in DARA-induced antibody-dependent cell cytotoxicity (ADCC).We have tested two MM (MM1.S, L-363) and seven AML (HL-60, U-937, THP-1, Molm-13, UOC-M1, OCI-AML2, Kasumi-1) cell lines representative of various levels of spontaneous CD38 cell surface density, as measured in thousands of CD38 molecules per cell by fluorescent anti-CD38 labeling (HIT2 clone) followed by quantitative flow cytometry using Cellquant calibrator beads (BioCytex). They ranged from very high CD38-positive (MM.1S, 51.0 ± 1.6 and UOC-M1, 46.5 ± 4.9) to high (OCI-AML2, 16.6 ± 1.5), low (L-363, 7.1 ± 0.1 and THP-1, 7.0 ± 0.2), very low (Molm-13, 2.1 ± 1.0 and Kasumi-1, 1.1 ± 1.2) and CD38-negative (HL-60, U-937). After exposure to 10 nM INE or vehicle for 72h, DARA-induced ADCC was assessed using either the calcein staining of target cells exposed to healthy donors peripheral blood mononuclear cells (PBMC) as effector cells, or the ADCC Reporter Bioassays Kit from Promega (G7018).After control vehicle incubation, DARA-induced ADCC was concentration-related in the range of 0.01 to 1 µg/mL in all cell lines and achieved a maximum ranging from 7% in THP-1, although a CD38-negative cell line, to respectively 27% and 28% in MM1.S and UOC-M1, the two highest CD38-positive cell lines. However, the correlation between maximal DARA-induced ADCC and CD38 surface density did not reach statistical significance (Spearman's rank correlation r = 0.6134, p = 0.09, n = 9).After incubation with 10 nM INE, the initially CD38-negative U-937 and HL-60 cells were found to express low (7.9 ± 0.3) and high (25.6 ± 7.3) surface CD38 levels, respectively. All the CD38-positive cell lines have shown a marked increase in the number of surface CD38 molecules: MM1.S (228 ± 12), OCI-AML2 (76,7 ± 6.5), UOC-M1 (68.8 ± 3.1), L-363 (63.9 ± 1.6), Molm-13 (38.2 ± 8.9), THP-1 (17.9 ± 0.9) and Kasumi-1 (15.7 ± 3.3) (paired Student's t-test, p = 0.02, n = 7). DARA-induced ADCC was concentration-related in the same 0.01 to 1 µg/mL range. Maximal DARA-induced ADCC was increased by 10 nM INE in all cell lines (paired Student t-test, p = 0.0025, n = 9), ranged from 20% in the lowest CD38-expressing cell line U-937 to 42% in the highest CD38-expressing cell line MM1.S, and was significantly correlated with the number of CD38 molecules/cell (Spearman's rank correlation r = 0.8333, p < 0.01, n = 9).Altogether, these results suggest that inecalcitol could represent a therapeutic agent of choice to enhance the clinical response of MM and AML patients to anti-CD38 antibodies such as daratumumab. DisclosuresMouly:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Rousseau:Hybrigenics: Employment. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

CPX-351: changing the landscape of treatment for patients with secondary acute myeloid leukemia.

Multiple novel therapeutic agents against acute myeloid leukemia (AML) have been evaluated in the past several decades without meaningful clinical improvement in outcomes, especially for AML patients age ≥60, where the overall incidence of AML is highest. Therapeutic options mainly consist of hypomethylating agents, ongoing clinical trials and, less commonly, intensive cytotoxic chemotherapy. CPX-351, a novel liposomal formulation which encapsulates cytarabine and daunorubicin in 5:1 molar ratio, has shown promising efficacy, leading to recent US FDA approval for front-line therapy for patients with therapy-related AML and AML with myelodysplasia-related changes based on a large multicenter Phase III clinical trial. This review summarizes the clinical development of CPX-351 as induction therapy.

All-trans retinoic acid enhances cytotoxic effect of T cells with an anti-CD38 chimeric antigen receptor in acute myeloid leukemia.

We reported that T cells with anti-CD38-chimeric antigen receptors (CAR) eliminated B-cell lymphoma cells expressing CD38. To employ anti-CD38-CAR against acute myeloid leukemia (AML) blasts not expressing CD38, it is necessary to induce or increase the intensity of CD38 expression. A lactate dehydrogenase (LDH)-releasing assay and flow cytometry showed that anti-CD38-CAR T cells were cytotoxic against AML lines (THP-1 and CMK) expressing high CD38 levels (>99%), in time- and number of effector-dependent manners. In other AML lines (KG1, U937 and HL60) partially expressing CD38, CD38+ AML cells were killed by CD38-specific T cells, but CD38− AML cells remained survived. Intriguingly, 10 nM all-trans retinoic acid (ATRA) augmented CD38 expression in KG1, U937 and HL60 cells and primary leukemic cells from AML patients. Moreover, the withdrawal of ATRA from the medium decreased CD38 expression in AML cells. Killing effects of anti-CD38-CAR T cells against AML lines and AML cells were limited without ATRA, whereas CD38-specific T cells enhanced cytotoxicity on AML cells by ATRA in association with enhanced CD38 expression. These results indicate that anti-CD38-CAR T cells eliminate AML cells through CD38 expression induced by ATRA.

Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks

ABSTRACTHistone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.

Alloreactive Natural Killer Cells for the Treatment of Acute Myeloid Leukemia: From Stem Cell Transplantation to Adoptive Immunotherapy.

Natural killer (NK) cells express activating and inhibitory receptors, which recognize MHC class-I alleles, termed “Killer cell Immunoglobulin-like Receptors” (KIRs). Preclinical and clinical data from haploidentical T-cell-depleted stem cell transplantation have demonstrated that alloreactive KIR-L mismatched NK cells play a major role as effectors against acute myeloid leukemia (AML). Outside the transplantation setting, several reports have proven the safety and feasibility of NK cell infusion in AML patients and, in some cases, provided evidence that transferred NK cells are functionally alloreactive and may have a role in disease control. The aim of the present work is to briefly summarize the most recent advances in the field by moving from the first preclinical and clinical demonstration of donor NK alloreactivity in the transplantation setting to the most recent attempts at exploiting the use of alloreactive NK cell infusion as a means of adoptive immunotherapy against AML. Altogether, these data highlight the pivotal role of NK cells for the development of novel immunological approaches in the clinical management of AML.

The Antibody-Drug Conjugate (ADC) IMGN779 Is Highly Active in Vitro and in Vivo Against Acute Myeloid Leukemia (AML) with FLT3-ITD Mutations

IMGN779 is a CD33-targeted ADC utilizing DGN462, a novel DNA-alkylating agent consisting of an indolino-benzodiazepine dimer containing a mono-imine moiety. CD33 is expressed on the surface of about 90% of AML cases, with elevated levels of CD33 found in cases having molecular markers associated with poor prognosis, including mutations in FMS-like tyrosine kinase 3 (FLT3). The internal tandem duplication mutation (FLT3-ITD) is the most common FLT3 mutation, present in about 20-25% of AML cases. Patients with FLT3-ITD AML have a worse prognosis than those with wild-type (WT) FLT3, with an increased rate of relapse and a shorter duration of response to induction chemotherapy.IMGN779 was found to demonstrate targeted activity against AML cell lines in vitro, with IC50 values ranging from 2-3,000 pM. The MV4-11 cell line, which has a FLT3-ITD mutation, was the most sensitive to IMGN779 of the cell lines tested, with an IC50 of 2 pM. We evaluated the in vivo activity of IMGN779 against MV4-11 xenografts in SCID mice; IMGN779 was highly active (T/C = 1 %) at a single dose of 0.6 mg/kg (conjugate dose, 10 µg/kg DGN462 dose), resulting in complete tumor regressions (CR) in 3/6 animals and partial regressions (PR) in 6/6 animals. A DGN462-ADC to a non-relevant target was inactive (T/C = 95%) at the same dose, demonstrating that the activity of IMGN779 was due to its CD33 targeting. IMGN779 has previously been shown to be highly active against AML xenograft models without FLT3-ITD mutations, at minimally efficacious doses of 0.6 mg/kg (10 µg/kg DGN462), demonstrating that the presence of FLT3-ITD does not confer resistance to IMGN779 treatment.IMGN779 was also highly active in vitro against primary patient AML cells isolated from peripheral blood or bone marrow samples. Patient AML cells with FLT3-ITD were more sensitive to IMGN779 compared with FLT3 WT AML samples. IC50 values in FLT3-ITD samples ranged from 10 to 300 pM. CD33 expression was generally greater on FLT3-ITD leukemic blast cells than on FLT3 WT blasts, which likely contributed to their increased sensitivity to IMGN779. In long term cultures, IMGN779 showed a dose dependent decrease in leukemic stem cell (LSC) colony formation using an AML patient sample with both FLT3-ITD and NPM1 mutations, which are an even worse prognostic marker than FLT3-ITD alone. In contrast, colony formation increased in normal bone marrow, indicating that normal hematopoietic stem cells (HSCs) were spared.The differential expression of CD33 on LSC compared to HSCs makes CD33 an attractive target for treatment of AML, with the potential to eliminate LSCs and, thus, minimal residual disease in FLT3-ITD AML. The potent in vitro activity of IMGN779 against FLT3-ITD AML cell lines and primary patient FLT3-ITD AML progenitor cells and LSCs and its high level of CD33-targeted in vivo activity in a FLT3-ITD AML xenograft model support the advancement of IMGN779 as a potential treatment for AML, including FLT3-ITD AML. DisclosuresWhiteman:ImmunoGen, Inc.: Employment. Noordhuis:ImmunoGen, Inc.: Research Funding. Walker:ImmunoGen, Inc.: Employment. Watkins:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Johnson:ImmunoGen, Inc.: Employment. Schuurhuis:ImmunoGen, Inc.: Research Funding. Ossenkoppele:ImmunoGen, Inc.: Research Funding. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

Glucopsychosine increases cytosolic calcium to induce calpain-mediated apoptosis of acute myeloid leukemia cells

To identify novel anti-cancer agents, we created and screened a unique nutraceutical library for activity against acute myeloid leukemia (AML) cells. From this screen, we determined that glucopsychosine was selectively toxic toward AML cell lines and primary AML patient samples with no effect toward normal hematopoietic cells. It delayed tumor growth and reduced tumor weights in mouse xenograft models without imparting toxicity. Glucopsychosine increased cytosolic calcium and induced apoptosis through calpain enzymes. Extracellular calcium was functionally important for glucopsychosine-induced AML cell death and surface calcium channel expression is altered in AML cells highlighting a unique mechanism of glucopsychosine's selectivity.

Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common ‘core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain ‘super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.