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The treatment landscape for acute myeloid leukemia has expanded significantly in the past 5 years with the approval of several therapeutic small molecules. While agents such as FLT3 inhibitors and IDH inhibitors are restricted for patients with specific mutations, the selective BCL-2 inhibitor venetoclax combined with a hypomethylating agent or low-dose cytarabine was approved after demonstrating frontline efficacy across a molecularly heterogenous group of patients. Currently, venetoclax is being investigated in combination with multiple other therapies as the role of the intrinsic apoptotic pathway in acute myeloid leukemia continues to be explored.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy arising from the aberrant transformation of plasmacytoid dendritic cells (pDCs) and involving skin, bone marrow, lymph nodes, and central nervous system. Characteristically unique from other myeloid neoplasms, BPDCN cells express CD4, CD56, and CD123 as well as TCL-1 and TCF4 in almost all cases. Historically, this malignancy has exhibited a poor prognosis, with median survival of less than 2 years. Traditional treatment approaches have involved conventional cytotoxic chemotherapy followed by hematopoietic stem cell transplantation; however, patients frequently relapse with chemotherapy-resistant disease. We have recently entered a modern era of therapy with targeting of CD123, with first-in-class agent tagraxofusp, a CD123- targeted agent approved by the US Food and Drug Administration for therapy of patients with BPDCN ages 2 and older. Relapsed and refractory BPDCN remains an elusive therapeutic challenge, but better understanding of the underlying pathophysiology has led to the development of other CD123-targeted agents and combination therapy, as well as agents targeting beyond CD123. Specifically, the use of venetoclax in targeting BCL2 has been promising in BPDCN treatment. This review will focus on the underlying diagnostic markers of BPDCN which have led to novel targeted treatment strategies, as well as future directions in therapy we can expect in coming years.

Introduction BCL-2 inhibition has transformed the therapeutic landscape of acute myeloid leukemia (AML). However, 10–50% of newly diagnosed patients with AML may not respond to venetoclax and HMA or LDAC, and 3–15% patients may not respond to venetoclax with intensive or non-intensive chemotherapy.1–6 In addition, up to 40% of responding patients may relapse with low rates of response of 20% to salvage therapy and poor overall survival of 2 months after relapse.7 Clinical and biological factors associated with primary and acquired resistance to venetoclax include secondary AML, monocytic differentiation, complex cytogenetics, mutations in TP53, BAX, dependence on other anti-apoptotic proteins, altered metabolism of nicotinamide, fatty acids, and oxidative phosphortylation.3,8–14

The treatment landscape for AML has grown significantly in the past 5 years with the approval of several therapeutic small molecules. While agents such as FLT3 inhibitors and IDH inhibitors are restricted for patients with specific mutations, the selective BCL-2 inhibitor venetoclax combined with a hypomethylating agent (HMA) or low-dose cytarabine (LDAC) was approved after demonstrating frontline efficacy across a molecularly heterogenous group of patients. Currently, venetoclax is being investigated in combination with multiple other therapies as the role of the intrinsic apoptotic pathway in AML continues to be explored.

Background DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. Method We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. Results Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells’ metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells’ ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. Conclusion In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.

Introduction: Recent FDA approvals of novel therapies and hypomethylating agents/venetoclax (HMA-ven) has transformed AML therapy. CR with partial hematological recovery (CRh), defined as bone marrow blasts 0.5×109/L, and platelet count >50×109/L, was used as a surrogate marker for efficacy due to limited data on HMA-ven. We evaluated the impact of CRh on pts treated with 10-day decitabine and venetoclax (DEC10-VEN, NCT03404193). Method: Patients with newly diagnosed AML aged >60 years or patients with secondary AML and antecedent hematological disorder were included. Decitabine 20 mg/m2 IV for 10 days with oral venetoclax 400 mg daily was administered for induction and decitabine (5 days) with daily venetoclax for consolidation. Responses initially recorded were complete response (CR), CR with incomplete hematologic recovery (CRi), and morphologically leukemia-free state (MLFS), according to ELN 2017 criteria. Pts who achieved CRi and MLFS were re-classified to CRh. Outcomes evaluated were overall survival (OS) and event-free survival (EFS). Full protocol and interim results have been published (DiNardo et al. Lancet Haematol. 2020;7:e724–36) Results: From January 2018 to February 2021, 125 patients (79 [63%] newly diagnosed, 46 [37%] secondary AML) with a median age of 71 yrs (range 48–89) were included. 69 (55%) pts achieved CR, 11 (8%) pts achieved CRh, 14 (11%) pts achieved CRi, 10 (8%) pts achieved MLFS, and 21 (17%) pts were refractory. Of the 11 CRh pts, 8 (73%) patients were previously classified as CRi and 3 (27%) as MLFS. Among pts achieving CR, the median OS was 24.5 mo, and median EFS was 18.0 mo. CRh was achieved with a median of 1 (range 1–3) cycles. Median OS of CRh vs CRi was 16.9 vs 5.8 mo (HR: 0.47, 95% CI: 0.19–1.13, p=.06), and CRh vs MLFS was 16.9 vs 6.2 mo (HR: 0.34, 95% CI: 0.13–0.93, p=.007). Median EFS of pts with CRh vs CRi was 14.1 vs 5.8 mo (HR: 0.49, 95%CI: 0.21–1.14, p=.08); CRh vs MLFS was 14.1 vs 6.2 mo (HR: 0.37, 95% CI: 0.14–1.01, p=.016). Pts achieving CRh had durable remission with a duration of response of 6.0 vs 2.5 mo with CRi and 1 mo with MLFS. Conclusion: In pts with newly diagnosed and secondary AML treated with DEC10-VEN, pts achieving CRh had a trend toward improved OS and EFS compared with CRi or MLFS.

Context: Oxidative phosphorylation (OxPhos) inhibition induces mitochondrial trafficking from BM stromal cells to AML and is accompanied by mitochondrial fission and mitophagy. The mitochondrial coupling and crosstalk with BM stromal cells facilitate the development of resistance to OxPhos inhibition in AML cells. Objective: Acute myeloid leukemia (AML) cells are highly dependent on OxPhos for survival and continually adapt to fluctuations in nutrient and oxygen availability in the environment. We investigated how the BM microenvironment affects the response to OxPhos inhibition in AML by using a novel complex I OxPhos inhibitor, IACS-010759. Design: Primary AML cells from 26 patients, primary normal MSCs, and AML cell lines were utilized and treated with the OxPhos inhibitor IACS-0107598 and/or cytarabine. Cap analysis of gene expression transcriptome analyses for AML PDXs, primary samples, and AML cell lines has been performed. We performed an extracellular flux assay to measure the oxygen consumption rate. To visualize mitochondria, AML cell lines and MSCs were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. Electron microscopy and immunoelectron microscopy analysis have been performed. Results: Cellular adhesion, growth, and apoptosis assays, along with measurements of mtDNA expression and mitochondrial reactive oxygen species generation, indicated that direct interactions with BM stromal cells triggered the compensatory activation of mitochondrial respiration and resistance to OxPhos inhibition in AML cells. Mechanistically, OxPhos inhibition induced (1) transfer of mesenchymal stem cell (MSC)-derived mitochondria to AML cells via tunneling nanotubes under direct-contact co-culture conditions and (2) mitochondrial fission with an increase in functional mitochondria and mitophagy in AML cells. Mitochondrial fission is known to enhance cell migration, and we observed mitochondrial transport to the leading edge of protrusions of migrating AML cells toward MSCs by electron microscopy analysis. We further demonstrated that cytarabine, a commonly used antileukemia agent, increased IACS-010759-triggered mitochondrial transfer from MSCs to AML cells. Conclusions: Our findings indicate an important role of exogenous mitochondrial trafficking from BM stromal cells to AML cells and of endogenous mitochondrial fission and mitophagy in the compensatory adaptation of leukemia cells to energetic stress in the BM microenvironment.

Context: BCL2A1 exerts a pro-survival function in FLT3-ITD/D835-double mutant acute myeloid leukemia (AML) cells and may be a putative novel therapeutic target in FLT3-mutant AML. The combination of gilteritinib, which reduced BCL2A1 with venetoclax, was highly synergistic in FLT3-ITD/D835-double mutant cells. Objective: While FLT3 tyrosine kinase inhibitors (TKI) emerged as a new therapeutic option in AML with internal tandem duplications in the juxtamembrane domain of FLT3 (FLT3-ITD), the prognosis of FLT3-mutated AML is still poor because heterogeneous clonal populations with additional acquired FLT3 and Ras mutations emerge and cause drug resistance. FLT3-ITD plus a point mutation at residue D835 in the tyrosine kinase domain (FLT3-ITD/D835) often cause resistance or decrease sensitivity to TKIs. In this study, we investigated new drug targets in AML with FLT3-ITD and TKD double mutations. Design: We carried out cap analysis of gene expression (CAGE) in primary AML cells from 26 patients to characterize transcriptome differences between FLT3-ITD-positive and FLT3-ITD/D835 double-positive primary AML cells. Selected genes were confirmed and validated with immunoblotting and RT-qPCR using FLT3-ITD-positive and FLT3-ITD/D835-positive MV4-11 cells. Furthermore, we generated FLT3-ITD-postive cells with overexpression of the selected genes using lentivirus vectors. Results: CAGE demonstrated that AML cells harboring FLT3-ITD/D835 double mutations expressed increased BCL2A1 gene transcripts compared to those with FLT3-ITD alone. RT-qPCR and immunoblot analysis also revealed that FLT3-ITD/D835-positive MV4-11 cells expressed higher transcript and protein levels of BCL2A1 than MV4-11 cells with FLT3-ITD mutation alone. Overexpression of BCL2A1 attenuated sensitivity to venetoclax and decreased sensitivity to quizartinib in AML cells with a heterozygous FLT3-ITD mutation (FLT3-wildtype/FLT3-ITD) but not in cells with a homozygous mutation (FLT3-ITD/FLT3-ITD). Gilteritinib targets BCL2A1 through inactivation of STAT5, and combinatorial gilteritinib and venetoclax were highly synergistic in FLT3-ITD/D835 double-positive cells. Furthermore, inhibition of BRD4 with the BET inhibitor CPI-0610 exhibited anti-tumor activity in FLT3-ITD/D835 double-positive AML and suppressed BCL2A1 expression in these cells. Conclusions: This study elucidates overexpression of BCL2A1 as a novel survival mechanism in FLT3-ITD/D835 mutant AML cells and identifies BCL2A1 as a putative novel therapeutic target in FLT3-mutant AML.

Context: Isocitrate dehydrogenase 2 (IDH2) mutations occur in 5% of patients (pts) with MDS. Enasidenib (ENA) is a selective oral mutant-IDH2 inhibitor with single-agent activity in relapsed/refractory AML. Objective: To evaluate the efficacy and tolerability of ENA, as monotherapy or in combination with azacitidine (AZA), in pts with higher-risk IDH2-mutated MDS (NCT03383575). Design and Setting: Multi-institutional, open-label, two-arm, non-randomized, phase II study. Patients and Interventions: Pts with higher-risk (R-IPSS > 3, or high-molecular-risk MDS/CMML, or RAEB-T MDS) enrolled in Arm A and received ENA100 mg orally daily for 14 or 28 d of each 28-d cycle + AZA 75 mg/m2 IV or SC on d 1–7 of each cycle (ENA+AZA). ENA duration was decreased to 14 d of each cycle, per amendment, to mitigate cytopenias. Pts with refractory or progressive MDS prior to HMA therapy enrolled in Arm B and received ENA alone (ENA), continuously, in 28-d cycles. Main Outcome Measures: The primary endpoint was overall response rate (ORR) [complete remission (CR), marrow CR (mCR), partial remission (PR), and hematologic improvement (HI)]. Other endpoints include safety and survival outcomes. Results: 48 pts received ENA+AZA (n = 26) or ENA (n = 22). The median age was 73 yrs (range, 46–83). Most pts (72%) had HMR: ASXL1 (39%) and RUNX1 (17%). Common Tx-related grade 3–4 AEs in the ENA+AZA arm were neutropenia (64%), thrombocytopenia (28%), and anemia (8%); these occurred in 10%, 0%, and 5%, in the ENA arm, respectively. IDH differentiation syndrome occurred in 3 pts (12%) in the ENA+AZA and 5 pts (24%) in the ENA arm. In response-evaluable pts (n = 46), ORR was 84% (n = 21/25; 24% CR + 8% PR+44% mCR+ 8% HI] in the treatment-naive Arm A and 43% (n = 9/21; 24% CR+5%PR+5% mCR+10% HI) in the HMA failure ENA arm B. After a median follow-up of 12.6 mo, median OS was 32.2 mo in the ENA+AZA arm and 21.3 mo in the ENA arm. Conclusions: ENA is well-tolerated and shows promising efficacy in IDH2-mutated higher-risk MDS. Follow-up and accrual are ongoing.

Background: De novo nucleotide synthesis is a dynamic process that can address the enormous demand for nucleotides and other macromolecules required in acute myeloid leukemia (AML) proliferation. Hence, we hypothesized that targeting de novo nucleotide synthesis would lead to the depletion of the nucleotide pool and pyrimidine starvation in leukemic cells compared to their non-malignant counterparts, impacting proliferative and differentiation pathways. Emvododstat (PTC299) is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis that is currently in a clinical trial for the treatment of AML. Objectives: The goals of these studies were to demonstrate that emvododstat effects leukemia growth due to the inhibition of de novo pyrimidine nucleotide synthesis. Comprehensive analyses of mitochondrial function, metabolic signaling in PI3K/AKT pathways, apoptotic signatures, and DNA damage responses were evaluated. The clinical relevance of emvododstat efficacy was confirmed in an AML-PDX model. Results: Emvododstat treatment in cytarabine-resistant AML cells and primary AML blasts induced apoptosis, differentiation, and reduced proliferation, with corresponding increases in annexin V- and CD14-positive cells. Indeed, the inhibition of de novo nucleotide synthesis compromises the dynamic metabolic landscape and mitochondrial function, as indicated by decreases in the oxygen consumption rate (OCR) and mitochondrial ROS/membrane potential. These effects can be reversed by the addition of exogenous uridine and orotate. Further immunoblotting and mass cytometry (CyTOF) analyses demonstrated changes in apoptotic and cell signaling proteins (cleaved PARP, cleaved caspase-3) and DNA damage responses (TP53, γH2AX) and PI3/AKT pathway downregulation in response to emvododstat. Finally, in a PDX mouse model of human AML, emvododstat treatment improved survival compared to mice treated with vehicle (median survival 40 days vs 30 days, P=0.0002). This corresponded with a reduction in the bone marrow burden of leukemia and increased expression of differentiation markers in mice treated with PTC299. Conclusion: Inhibition of de novo pyrimidine synthesis triggers differentiation, apoptosis, and/or inhibition of proliferation in AML models. Emvododstat is a novel dihydroorotate dehydrogenase inhibitor being tested in a clinical trial for the treatment of myeloid malignancies and COVID-19.

Context: Relapsed/refractory (R/R) acute myeloid leukemia (AML) has poor outcomes. While lower-intensity venetoclax-containing regimens are standard for older/unfit patients with newly diagnosed AML, it is unknown how such regimens compare to intensive chemotherapy (IC) for R/R AML. Objective: To compare outcomes of R/R AML treated with 10-day decitabine and venetoclax (DEC10-VEN; NCT03404193) vs IC-based regimens Design: This was a retrospective study. Propensity scores derived from baseline characteristics were used to match patients treated with DEC10-VEN and IC to minimize bias. Patients and Setting: Sixty-five patients in the DEC10-VEN cohort were matched to 130 IC recipients treated at the University of Texas MD Anderson Cancer Center. The median ages for the DEC10-VEN and IC groups were 64 and 58 years, respectively, and baseline characteristics were balanced between the two cohorts. Interventions: DEC10-VEN comprised of decitabine for 10-days and venetoclax 400 mg daily (DiNardo et al. Lancet Haematol. 2020.) IC-based regimens included idarubicin with cytarabine (IA), with or without cladribine (CLIA), clofarabine (CIA), or fludarabine (FIA or FLAG-IDA), with or without additional agents. Main Outcome Measures: Response rates per ELN 2017 criteria, rate of negative MRD by flow cytometry, early mortality, safety, event-free survival (EFS), and overall survival (OS). Results: DEC10-VEN conferred significantly higher responses compared to IC, including higher overall response rate (60% vs 36%, odds ratio [OR]: 3.28, p Conclusions: DEC10-VEN represents an appropriate salvage therapy and may offer better responses and survival compared to IC in adults with R/R AML. Funding: NCI CCSG CA016672, R01CA235622.

Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, hematologic malignancy, comprised of clonal plasmacytoid dendritic cells that are CD123-positive and associated with historically poor outcomes. In 2018, tagraxofusp (TAG), a targeted therapy directed to CD123, became the first-in-class FDA approved treatment for children and adults with BPDCN ages 2 years and older. Treatment with TAG resulted in durable clinical responses with a manageable safety profile in both treatment-naive and relapsed/refractory (R/R) patients. Herein, we present long-term follow-up results ( NCT02113982 ). Objectives To assess the safety and efficacy of TAG in patients with untreated and previously treated BPDCN. Methods In this multicenter, multi-stage, single-arm trial 44 patients with treatment-naive or R/R BPDCN were treated at the indicated dose (12 mcg/kg). In Stage 1 (dose escalation) patients received TAG daily IV infusions at 7 or 12 mcg/kg on days 1–5 of a 21-day cycle. In Stage 2 (expansion) and Stage 3 (pivotal, confirmatory), TAG was dosed at 12 mcg/kg. Key efficacy measures included objective response rate (ORR), complete response (CR), clinical CR (CRc), defined as CR with residual skin abnormality not indicative of active disease, and overall survival (OS). Results Twenty-nine treatment-naive and 15 R/R BPDCN patients were treated for a median duration of 5 and 3 cycles, respectively. Most common treatment emergent adverse events (TEAEs) include hypoalbuminemia, increased transaminases, thrombocytopenia, nausea, fatigue and pyrexia. The most serious adverse event was capillary leak syndrome (CLS), 19% in all grades (one patient with Grade 4 and two with Grade 5). Incidence of TEAEs leading to TAG discontinuation was 2%; temporary dose interruptions for TEAE management were common (68%). In treatment-naive patients ORR was 90%, CR+CRc rate was 72%, and 45% were bridged to stem cell transplant. Median time to response was rapid; 65% of patients achieved complete response by C2. Median overall survival was 26 months; 24-month survival probabilities of 52%. In R/R patients, ORR was 67%, including 2 CR+CRc. Conclusions In the largest prospectively designed study conducted to date, TAG demonstrated robust and durable responses with a median OS of 26 months and a well-characterized safety profile including recognition for CLS.

Context There are concerns that the use of immune checkpoint inhibitors (ICIs) after allogeneic stem cell transplant (allo-SCT) may increase the incidence of acute graft-versus-host disease (aGVHD). Objective We report our experience with ICIs used after allo-SCT in AML/MDS patients with a focus on the impact of post-transplant cyclophosphamide (PTCy). Methods We retrospectively analyzed 16 AML and 5 MDS patients who received nivolumab (n=16) or ipilimumab (n=5) for disease relapse after allo-SCT at our institution. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results Median age 54 years; 12 patients (57%) received PTCy. Transplantation characteristics were comparable between the two groups (PTCy vs no-PTCy), except for lower hematopoietic stem cell comorbidity index noted in the PTCy group (91% vs. 37.5%, P=.04). The PTCy group had shorter median time from allo-SCT to ICI initiation (5 vs. 26 months, P=.04). Over a median follow-up of 5.3 months, the frequency of grade 2–4 aGVHD was 19% in the entire cohort and was lower in PTCy group (16% vs 22%, P=0.7). All aGVHD cases were noted following nivolumab initiation. In patients who developed aGVHD, the median time from allo-SCT to ICI initiation was 4 months in PTCy group vs 9 months in the non-PTCy group (P=.17). Median time from ICI initiation to aGVHD was 24 days (range, 12–31) in the entire cohort and 22 days in PTCy group vs 16 days in non-PTCy (P=.31). After controlling for comorbidities and time from transplant to ICI initiation, we found that PTCy was significantly associated with a 90% reduced risk of aGVHD (HR= 0.1; 95% CI: 0.02–0.6; P=.01). No chronic GVHD or grade ≥2 immune-related adverse events were reported. The PTCy group had a significantly longer median PFS (22.4 vs. 5.2 months, P=.02) and a trend toward longer median OS (22.4 vs. 5.2 months, P=.08). Fourteen patients (67%) died, and no deaths were attributed to aGVHD. Conclusions The use of ICI for AML/MDS relapse after allo-SCT may be a safe and feasible option. PTCy appears to decrease the incidence of aGVHD in this cohort of patients.

Context Chemotherapy resistance is one of the leading causes of disease progression and relapse in patients with acute myeloid leukemia (AML). The interaction between mesenchymal stromal cells (MSCs) and AML cells in the bone marrow (BM) is critical for leukemogenesis and chemotherapy resistance. Objective As tumor initiation and chemotherapy resistance are characteristic features of leukemia stem cells, we hypothesize that BM-MSCs induce a stem-like phenotype in AML cells. The objectives of this study are to determine the effect of stromal cells on the stem cell function of AML cells and to identify signaling pathways that support AML-stroma interactions in the BM microenvironment. Design We cultured AML cell lines and patient samples with or without BM-MSCs and measured aldehyde dehydrogenase (ALDH) activity in AML cells by ALDEFLUOR™ assay. In addition, we performed genomic and proteomic analyses to identify signaling pathways that support AML-stroma interactions. Finally, we used specific inhibitors that target ALDH activity to sensitize AML cells to chemotherapeutic agents. Results Co-culture of AML cells with BM-MSCs significantly induced ALDH activity in AML cells, mainly through upregulation of ALDH2 and ALDH1L2 isoforms. TCGA data analysis revealed that ALDH2 is a prognostic factor in AML. Mechanistic studies revealed that TGF-β1-regulated gene signature is activated in AML cells co-cultured with stromal cells. Knockdown of TGF-β1 in BM-MSCs inhibited stroma-induced ALDH activity and ALDH2 expression in AML cells, whereas treatment with recombinant TGF-β1 induced an ALDH+ phenotype in AML cells. We also found that the non-canonical TGF-β pathway, through p38 activation, regulates ALDH2 expression in AML cells. Pharmacologic inhibition of ALDH2 significantly inhibited BM-MSC-induced ALDH activity and sensitized AML cells to chemotherapy. Conclusion BM-MSCs induce ALDH+ stem cell phenotype in AML cells through the non-canonical TGF-β pathway. ALDH2 is the crucial isoform regulating ALDH activity in AML cells and is a prognostic factor. Pharmacologic inhibition of ALDH2 sensitizes AML cells to standard chemotherapy. Grant Acknowledgement This study is partially supported by Leukemia SPORE CDA, IRG from MDACC, Cure Sonia Foundation and Golfer's Against Cancer to VLB. NIH (CA055164), MDACC Support Grant (CA016672), CPRIT-MIRA, and Paul and Mary Haas Chair in Genetics to MA.

Context IMGN632, a novel CD123-targeting ADC, demonstrates a favorable safety profile and complete remissions as a monotherapy in patients with relapsed/refractory AML and BPDCN ( NCT03386513 ). In pre-clinical models, IMGN632 has been shown to be synergistic with venetoclax (BCL-2 inhibitor) (EHA, 2019, abstract #PF201). Objective Given the recent approval of the combination of azacitidine (AZA, a hypomethylating agent) and venetoclax (VEN) in AML patients unfit for intensive chemotherapy, we investigated the combination of IMGN632 with AZA and the triple combination of IMGN632, AZA, and VEN in preclinical models. Results IMGN632, AZA, and VEN were highly active and prolonged survival in a cell line-derived AML model and patient-derived AML xenograft (PDX) models. In EOL-1, treatment with the triple combination IMGN632+AZA+VEN was the most active and resulted in 100% reduction in tumor growth inhibition (T/C) when compared to Vehicle, IMGN632, and AZA+VEN treatments. In PDX 4023126, the triple combination group survived the longest and was highly active with 74-day tumor growth delay (T-C) and 128% increased life span (% ILS) demonstrating highest survival benefit over IMGN632 (33-day T-C; 57% ILS), and AZA+VEN (52-day T-C; 90% ILS). In PDX model, 4079574, the triple combination was highly active (62-day T-C; 105% ILS) compared to both IMGN632 alone (15-day T-C; 25.4% ILS) and VEN+AZA (1-day T-C; 1.7% ILS). In model 3912018, VEN+AZA (2-day T-C; 3% ILS) was not very active, and strikingly, the triple combination (231-day T-C; 335% ILS) was highly active and prolonged survival the most compared to IMGN632 alone (190-day T-C; 275% ILS) with all the mice surviving for >300 days. Conclusion The addition of IMGN632 to AZA+VEN in multiple AML xenograft and PDX models led to improved survival. These data support the addition of a CD123-targeted ADC with a novel DNA-damaging payload to standard of care, AZA+VEN in AML patients. The combination of IMGN632 and Venetoclax and/or Azacitidine is currently being tested in a Phase1b/2 clinical trial ( NCT04086264 ).

Context The combination of tyrosine kinase inhibitors (TKI) with intensive chemotherapy improves outcome of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-pos ALL), particularly among patients in molecular response within three months of therapy. Objective The aim of this study is to identify prognostic factors for relapse and/or death in patients with Ph-pos ALL after 3 months of therapy. Design Retrospective analysis of patients who enrolled on prospective clinical trials of the combination of hyper-CVAD (HCVAD) with a TKI. Setting MD Anderson Cancer Center. Patients or other participants We analyzed 204 patients with newly diagnosed Ph-pos ALL who were treated with HCVAD with a TKI; imatinib (N=44), dasatinib (N=88), and ponatinib (N=72). Interventions Molecular response was assessed at CR, at 3 months, every 3 months during the first 2 years, and every 3-6 months thereafter. Allogeneic stem cell transplantation was offered at the discretion of patient and treating physician. Ponatinib dose was reduced to 15 mg daily, once CMR is achieved. Main outcome measures Progression -free survival (PFS) was calculated from the date of treatment initiation until date of relapse, or death. Overall survival (OS) was calculated from the time of treatment initiation until death. Results By multivariate analysis for PFS and OS in patients who achieved CMR within 3 months of therapy, ponatinib therapy was the only significant independent factor predicting for progression (P=0.028; hazard ratio [HR], 0.388; 95% confidence interval [CI], 0.166-0.904) and death (P=0.042; HR, 0.379; 95% CI, 0.149-0.966); among patients who did not achieve CMR within 3 months, the achievement of major molecular response was a favorable prognostic factor. Though the number of patients with MMR and without molecular response (i.e., no CMR/MMR) is limited, there was a tendency of improved PFS in patients who received allogeneic stem cell transplant. Conclusions The achievement of CMR within 3 months of therapy is the landmark for long-term survival. Ponatinib maintains CMR, and prevents relapse and death in patients with newly diagnosed Ph-pos ALL. MMR within 3 months was associated with improved survival compared to the lack of MMR within 3 months of TKI therapy.

The human homologue of murine double minute 2 (HDM2) and HDM4 negatively regulate p53. HDM4 has not been assessed in acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We examined the expression of HDM4 and the short splicing variant, HDM4-S, in bone marrow samples obtained from 85 and 23 patients with AML and MDS, respectively, and 18 negative tumor staging bone marrow samples (used as the control). Immunohistochemical staining showed that HDM4 was overexpressed in 78 AML cases (92%) and 12 MDS cases (52%) compared with 1 stressed bone marrow sample (6%). Quantitative reverse transcriptase-polymerase chain reaction analysis of 8 AML and 11 low-grade (LG)-MDS cases confirmed that HDM4 and HDM4-S mRNA expression were also elevated in all AML cases. HDM4 and HDM4-S mRNA expression was elevated in 3 (27%) and 10 (91%) LG-MDS cases, respectively. HDM4 and HDM4-S mRNA levels were higher in those with AML than in those with LG-MDS. In leukemia cell lines, HEL and U937 predominantly expressed HDM4-S. In contrast, NALM6 expressed HDM4 and HDM4-S. Downregulation of HDM4 expression by treatment with small interfering RNA in NALM6 and HEL cells induced p21 expression but not increased apoptotic activity. Our results indicate that HDM4 is a potential therapeutic target in patients with AML or MDS.

Context Limited data are available on the outcome of adults with relapsed-refractory (R/R) T-cell acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). Objective We aimed to identify factors predictive for long-term survival and to establish the benchmark for expected outcomes in this setting. Main outcomes measured We reviewed characteristics and outcome of adults with R/R T-cell ALL or LBL treated at our institution between 2009 and 2019. We assessed response to salvage therapy and overall survival. To determine prognostic factors for survival, we performed univariate and multivariate analyses using the following baseline characteristics: age, disease type (ALL versus LBL), immunophenotypic subtype (early T-cell precursor [ETP] versus non-ETP), complex cytogenetics, central nervous system involvement at salvage, first complete remission (CR1) duration Results We identified 82 patients, including 39% in S1, 27% in S2, and 34% in S3+. ETP subtype was present in 35% of cases, and 16% had complex cytogenetics. Salvage therapy included standard-of-care regimens in 48 patients (59%), including nelarabine-based therapy in 21 patients (26%), pediatric-inspired asparaginase-based therapy in 11 patients (13%), clofarabine-based therapy in 9 patients (11%), and investigational-based therapies in 34 patients (41%), mainly venetoclax-based (18 patients; 22%). The CR/complete remission with incomplete hematological recovery (CRi) rate was 46%, with a median duration of response of 4 months. With a median follow-up of 37 months, the median OS was 8 months, and the 2-year OS was 23%. Only 14 patients were alive in remission at their last follow-up. In multivariate analysis, earlier salvage therapy was the only predictor for better survival (hazard ratio 0.57 [95% confidence interval, 0.33-0.99], P=0.049). A 3-month landmark analysis showed a survival benefit for consolidative hematopoietic stem cell transplant (HSCT) after CR to salvage therapy (2-year OS rate of 56% versus 24%, P=0.002). Conclusions The current study highlights the continuous unmet need of patients with R/R T-cell ALL treated with contemporary standard regimens and provides a reference for future clinical trials in this population.

Aims We evaluated efficacy of Azacitidine (AZA)+Venetoclax (VEN) combination vs. AZA+Placebo (PBO) in treatment-naive patients with acute myeloid leukemia (AML) ineligible for intensive therapy. Methods This phase 3, randomized, double-blinded, multicenter, placebo-controlled study included AML patients ineligible for intensive induction therapy due to medical comorbidities and/or age > 75 years. Patients were randomized 2:1 to either AZA+VEN(AZA: 75 mg/m2 subcutaneous or intravenous, days [d] 1-7 per 28-day cycle; VEN: 400 mg orally QD, d1-28 with 3-day ramp up in cycle 1) or AZA+PBO (PBO: orally QD, d1–28). Primary endpoint was overall survival (OS). Secondary endpoints were rates of: composite complete remission [complete remission (CR) + CR with incomplete count recovery (CRi)], CR+CRi by initiation of cycle 2, CR, transfusion independence, CR+CRi and OS by molecular subgroups, and event-free survival (EFS). A sample size of 400 was estimated to detect a hazard ratio (HR) of 0.7 in OS with 2-sided alpha of 4% and power of 87%. OS and EFS were analyzed by the Kaplan-Meier method and compared between arms using the log-rank test stratified by age (18 to Results As of 01/04/2020, 431 patients (median age 76 years, range 49-91) were randomized to AZA+VEN (n=286) or AZA+PBO (n=145). With median follow-up of 20.5 months (mos), median OS was 14.7 mos in AZA+VEN and 9.6 in AZA+PBO (HR: 0.66, 95% CI: 0.52–0.85, p Conclusions Among treatment-naive predominantly elderly patients with AML ineligible for intensive therapy, combination AZA+VEN showed statistically significant and clinically meaningful improvement in response rates and OS vs AZA, with a manageable safety profile. Abstract was previously published at EHA25.

Context We have reported that AML cells induce osteogenic differentiation in MSCs to facilitate AML growth in the bone marrow (BM) (Battula et al. JCI Insight, 2017). However, the effect of AML cells on various osteo-lineage cells in the BM microenvironment is not known. Objective i) To assess the stage-specific subsets of osteoblast lineage induced by AML cells in BME. ii) To investigate the effect of new bone formation using AML patient-derived-xenograft (PDX) mouse model. Design In this study, we used inducible triple transgenic mice with osterix (Osx) or osteocalcin (Ocn) reporter genes with genotype Osx-CreERt2; Ocn-GFP; ROSA-tdTomato to determine the effect of AML cells on various osteo-lineage cells. In addition, we used AML-PDX mouse models to investigate the impact of AML cell growth on new bone formation. Results In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osx+ cells, but no significant difference was observed in Ocn+ cells, suggesting AML cells expand osteoprogenitor cells but not mature osteoblasts in the BM. To determine whether the AML growth in the BM is associated with bone volume, we implanted AML-PDX cells (total 10 models) into NSG mice and analyzed femurs by micro-CT and histological analysis. Interestingly, we observed a dramatic increase in cortical bone thickness and new medullary bone formation in ∼50% of the PDX models tested. Interestingly, we found that increase in bone volume is associated with less aggressive PDX models (which takes 4-5 months to reach 90% PB engraftment). Aggressive PDX models (takes only 3-5 weeks to reach 90% PB engraftment) did not develop new bone. Masson-Goldner's trichrome staining confirmed weblike trabecular bone formation in mice with less aggressive PDX models, which was not observed in aggressive AML PDX models. Conclusion Our data suggest that AML cells induce supportive osteoprogenitor-rich niche in the BM. New bone formation in less aggressive AML-PDX models, but not in aggressive models suggests two different molecular mechanisms regulating AML growth in these two models. Finally, the role of osteoprogenitor niche in AML progression needs further investigation. Grant Acknowledgment Leukemia SPORE CDP, Emerson Collective, Golfers Against Cancer, LLS-TRP to VLB. RP to VLB.

Background Acute myeloid leukemia (AML) is a hematopoietic malignancy with an increased incidence among older patients. The long-term survival of older AML patients treated with established therapies remains poor. The BCL-2 inhibitor venetoclax with hypomethylating agent (HMA) improved response rates (CR/CRi> 65%) in newly diagnosed older AML patients, with median response duration of 11.3 months. Increased expression of program death-1 (PD-1) on T-cells is associated with AML immune-suppression, which contributes to chemoresistance. Venetoclax preserves T-cells immunity by sparing central memory T-cells and enhances anti-PD-1 efficacy in immune-competent mouse models in vivo. These findings prompted us to test the hypothesis that reactivation of immune function by PD-1 inhibition facilitates responses to venetoclax and decitabine. Results We examined serial peripheral blood (PB) samples from patients (n= 6) before and after decitabine and venetoclax (Dec_Ven, NCT03404193 ). The combination of Dec_Ven effectively reduced CD33+/CD34+ cells and increased CD3+ T-cells. The reduction of CD33+/CD34+ cells positively correlated with the depletion of circulating blasts (R = 0.64, p = 0.0001). Dec_Ven rapidly (Days 1–3) activated T-cells by upregulating CD69 and PD-1 in both CD4 and CD8 cells but led to T-cells exhaustion with persistently upregulated PD-1 and loss of CD69 on Days 3–8. Mass cytometry CyTOF immune profiling demonstrated that Dec_Ven reduced naive T-cells (CD45RA+CCR7+) but spared or increased frequencies of the central (CD45RA-CCR7+) and effector memory (CD45RA-CCR7-) CD4 or CD8 cells. In parallel, serum cytokine levels of IL2, IL-6, and INFg were increased with Dec_Ven therapy. To test if targeting PD-1 can amplify the anti-leukemia effect, PB cells from newly diagnosed AML patient pre- and post Dec_Ven were treated with anti-CD3 and anti-PD-1 in vitro. Blockade of PD-1 reduced CD33+/CD34+ cells in baseline sample (67% vs. 34%) and synergistically eliminated the residual leukemia cells in sample obtained on Day 3 following Dec_Ven therapy (4.5% post-Dec_Ven alone vs. 1.9% with anti-CD3/PD-1). Conclusions Our data indicate that combination Dec_Ven induces T-cells exhaustion but selectively preserves effector T-cells immune subsets. The “triple” combination therapy of HMA, venetoclax, and PD-1 antagonist may increase durability of responses by enhancing T-cell function and is being tested in clinical trial NCT02397720 . Funding This work is supported by R01CA235622.