Acute Myeloid Leukemia Therapy Research Articles

Chimeric Antigen Receptor-Engineered NK Cells Overcome CD226 Downregulation As an Immune Escape Mechanism in Acute Myeloid Leukemia

Background: Natural killer (NK) cells are part of the innate immune system, and their killing capacity depends on the inputs from activating and inhibitory receptors. CD226 is an activating receptor that plays a role in the immune surveillance of acute myeloid leukemia (AML). AML cells have developed mechanisms to escape NK cell cytotoxicity including downregulating activating receptors such as CD226 on the surface of NK cells. Chimeric antigen receptors (CARs) are synthetic receptors integrated into NK cells to direct their anti-leukemia activity, and CD38 is a potential target for NK CAR-based cellular therapy against AML. Understanding the dynamics between the endogenous activating and inhibitory NK cell receptors (such as CD226) and the introduced CAR can determine the development of more effective NK CAR-based therapy for AML. We hypothesize that CD38-directed CAR NK cells may overcome CD226 downregulation as a mechanism of immune escape in AML. Purpose: we aimed to investigate (1) the mechanisms developed by AML cells to escape recognition by the activating receptor CD226, (2) whether and how anti-CD38 CAR-armed NK cells can overcome AML immune escape mechanisms. Methods: we performed multiparametric mass spectrometry to characterize changes in CD226 expression in NK cells of individuals with AML. Then, we investigated CD226 expression in NK cell-AML cocultures to characterize CD226-related mechanisms by imaging flow cytometry. Finally, to explore the impact of CD226 knockout (KO) together with the role of CD38 CAR in NK cell functionality, we retrovirally transduced CD38 CAR combined with CRISPR-Cas9 CD226 KO followed by short- and long-term cytotoxicity assays, cytokine production techniques and immune synapse assays by imaging flow cytometry. Results: NK cells from AML patients expressed low levels of CD226 with impaired cytotoxicity compared to heathy donor NK cells. Co-culture of healthy donor NK cells with AML blasts resulted in CD226 downregulation secondary to its internalization and was dependent on expression of the CD226 ligand CD155 in AML cells. CD226 KO diminished NK cell cytokine production and decreased their killing capacity against AML cells. In contrast, CD38-targeting NK cells displayed enhanced and specific cytotoxic activity against primary AML cells. When we combined the CD38 CAR with a CD226 KO in NK cells, these cells maintained their activating potential, cytokine secretion levels, and anti-leukemic cytotoxicity (Figure 1). CD38 CAR / CD226 KO NK cells could build strong immune synapses (Figure 2) even in the absence of LFA-1, an integrin activated by CD226 signaling and essential for NK cell immune synapse formation, suggesting that the CAR molecule compensates for the absence of CD266-mediated LFA signaling. Furthermore, CD38 CAR NK cells were capable of forming immune synapses, that were at least partially independent from LFA-1. Conclusion: CAR-directed NK cells can surpass resistance mechanisms in AML, such as CD226 downregulation and dampening of LFA-1 recruitment into the immunological synapse. Thus, NK cell activation by artificially introduced and germline-encoded receptors can overcome distinct tumor-resistance mechanisms, CAR-driven NK cells being a potent option for the treatment of AML.

Concurrent Versus Sequential or No Triazole Anti-Fungal Therapy in Patients Undergoing 7+3 Plus Midostaurin Induction for FLT-3 Acute Myelogenous Leukemia

Background Midostaurin is a multikinase inhibitor approved for the treatment of adult patients with newly diagnosed FMS-like tyrosine kinase 3 (FLT3) mutated acute myeloid leukemia (AML) (Stone RM et al. NEJM 2017). Azole antifungal medications are commonly used in AML and are known to interact with anti-cancer drugs such as Midostaurin through the CYP3A pathway. This could result in an increase in midostaurin concentrations and toxicities, and lead to potential dose interruptions or modifications. However, the literature is scarce on outcomes in patients who are receiving an azole antifungal with intensive induction chemotherapy in combination with midostaurin (Sechaud R et al. Cancer Chemother Pharmacol 2022). In addition to the scarcity of literature, there are no midostaurin related dose modifications that are recommended with strong CYP3A inhibitors. The primary objective of this retrospective study was to compare induction efficacy and safety outcomes in patients receiving concurrent azole antifungal to patients receiving sequential or no azole antifungal therapy. Methods: From January 1, 2017, to January 30, 2023, we retrospectively reviewed all adult patients (18 or older) with newly diagnosed AML who received midostaurin in combination with cytarabine and an anthracycline (7+3). Concurrent azole therapy was defined as patients receiving an azole antifungal therapy with midostaurin for more than 70% of the midostaurin doses. Sequential azole therapy was defined as patients receiving azole antifungal therapy with midostaurin for less than 70% of the doses or no azole antifungal therapy with midostaurin therapy. Response outcome CR and CR without hematological recovery (CRi) were defined per standard International Working Group (IWG) AML response criteria. Minimal residual disease (MRD) assessment was done on post-induction bone marrow aspirate using multiparametric flow cytometric assessment. Adverse events were graded using NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. We compared efficacy and safety outcomes in patients who received azole antifungals concurrently to those who did not receive an azole or received it sequentially to midostaurin for treatment of FLT3 AML. Mann-Whitney U test method was used to compare continuous variables between these two groups. Pearson Chi-square test or Fisher Exact test was used to compare categorical variables (count, rate or proportion) between these two groups. The choice of concurrent azole or sequential azole antifungal was determined by physician choice. Results: There were 40 patients treated with 7+3+midostaurin, 18 of whom received concurrent azole therapy, while 22 received sequential or no azole therapy. There were no significant differences in baseline characteristics (Table 1) except for more patients receiving isavuconazole (moderate CYP 3A4 inhibitor) in the concurrent group (50% vs 0%; p<0.01) and more micafungin in the sequential or no azole group (0% vs 72%; p<0.01). Overall CR/CRi rate with concurrent versus sequential or no azole was 72% (13/18) vs 77% (17/22), with no significant difference in median days to ANC recovery (27 vs 29; p=0.98), platelet recovery (28 vs 32 days; p=0.67), and non-hematologic grade 3 toxicities (22% and 45%; p=0.13), respectively. There were higher, but not significant, rates of midostaurin dose reductions (6% vs 0%; p=0.26) and holds (17% vs 14%; p=0.79) in the concurrent azole group. Conclusion: In conclusion, the addition of azole antifungal was found to be equally safe and effective in the treatment of newly diagnosed FTL3 AML. Dose modifications in midostaurin appeared not to be necessary with concurrent azole antifungal therapy. Further analysis based on a larger cohort is needed to confirm the outcomes and safety endpoints reported in the current study. Given the importance of azole anti-fungal therapy in the treatment of AML and specifically in induction therapy for AML (Cornely et al, NEJM 2007), dose modifications for this drug interaction should be studied in the future to help guide clinicians on how to appropriately dose these drugs.

High Throughput Microfluidics Platform to Assess Synthetic Lethality and Novel Therapeutic Drug Combinations

Molecularly targeted therapies have reshaped cancer treatment in recent decades; however, not all patient cancers are eligible for or effectively treated by such approaches. In addition, resistance mechanisms (both pre-existing and acquired) limit clinical benefit. To these ends, combination therapies involving targeted agents are increasingly being developed for their potential to induce potent synthetic lethalities, bypass drug resistance, promote durable responses, limit adverse side effects via reduced dosing, and expand treatment options. In the context of acute myeloid leukemia (AML) specifically, several efficacious combination therapies have been approved for specific patient subsets, such as all-trans retinoic acid (ATRA) plus arsenic trioxide in the PML-RARA fusion acute promyelocytic subtype and Midostaurin plus Cytarabine and Daunorubicin in FLT3-mutant AML. Despite the availability of these and other therapies, there remains an unmet need for treatments against AML subtypes lacking actionable driver alterations or for which current therapies are insufficient. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with mixing two or more agents (e.g., screening all possible pairs of 100 drugs in a single cell line requires nearly 5,000 combination experiments). Thus, there is a practical need for methodologies that prioritize test compounds among broad candidate sets and high throughput technologies for rapid and efficient screening. We have developed a combination drug screening workflow that integrates machine learning-based predictions of potentially synergistic compound pairs with a droplet microfluidics-based screening platform. Our physical platform, termed the FlowMatrix, contains a series of nano wells arrayed in a 96 x 96 grid (for 9,216 total wells) in which cells are incubated and compounds delivered via emulsified droplets. To facilitate drug combination screening, the FlowMatrix is loaded column-wise with a 96-element library and subsequently loaded a second time along its rows with either the same or an independent drug library. In our current setup, one FlowMatrix can capture complete data for up to 100 unique drug pairs, which includes control, single-agent, and 4x4 dose combination measurements for each pair. Following multi-day incubation, viability for each well is assessed by the number of live cells imaged with fluorescence microscopy. Combinations with high scoring synergy based on Bliss independence criteria are then pushed to validation and further characterization pipelines. We are currently focused on identifying novel combination therapies for AML. Using small molecule libraries of diverse compound classes and machine learning to prioritize initial screening candidates, we have to-date profiled >8,500 AML cell line-drug combinations in our system. This encompasses >2,900 unique drug combinations profiled in 7 distinct AML cell lines. Among ~270 hits that emerged from these screens are established clinical and preclinical AML combination therapies, in addition to candidates that, to the best of our knowledge, are novel in the context of AML. Strong established hits in our screen include ATR inhibition plus Gemcitabine treatment as well as several combinations involving the BCL-2 inhibitor Venetoclax with chemotherapies (Decitabine and Daunorubicin), Quizartinib, Idasanutlin, and mTOR inhibitors. Thus, we have developed an efficient and cost-effective high throughput drug combinations profiling system that has uncovered candidate therapies that may expand treatment options for patients afflicted by AML.

Targeting Normal Myelopoiesis Negatively Affects CAR T Cell Activity

Chimeric antigen receptor (CAR) T cells have not been successful for the treatment of acute myeloid leukemia (AML) to date, and the reason for this is not clear. While CAR T cells have been traditionally studied as independent agents targeting tumor, in reality they work in the context of the endogenous host immune system, which may influence outcomes of therapy. We hypothesized that targeting normal myelopoiesis is detrimental to CAR T cell activity, and this creates a unique obstacle to effective CAR T cell therapy for AML. We generated prototypical CAR T cells targeting B cell (CD19) and myeloid (CD33) antigens, using a standard 2 nd generation CAR design with 4-1BB and CD3ζ costimulatory domains. In parallel we created tumor cell lines expressing both targets by adding CD33 expression to a CD19+ B cell line (Ramos +33), and by adding CD19 expression to a CD33+ myeloid cell line (THP1 +19). For ease of tracking tumor cells were labeled with GFP, and CAR T cells were labeled with RFP. CD19-targeting CAR T cells (CART19) and CD33-targeting CAR T cells (CART33) exhibited similar anti-tumor activity in standard in vitro cytotoxicity assays and in vivo NSG mouse models. We then proceeded to add PBMCs to CAR T cell and tumor co-culture and measured tumor cell clearance and CAR T cell expansion using flow cytometry and the Incucyte Live-Cell analysis system. Addition of PBMCs enhanced the tumor clearance and CAR T cell expansion of both CART19 and CART33 in short-term co-culture assays (7-10 days). Fractionation of PBMCs revealed that CD33+ classical monocytes were able to replicate the effect of PBMCs, while the presence of CD19+ B cells did not have any effect. Monocytes are viable in vitro for less than 7 days. To assess the long-term effects of monocytes on CAR T cells, we performed serial replating assays, in which fresh tumor cells and monocytes were added to CAR T cells every 7 days. Ongoing monocyte support further improved CART19 expansion and tumor clearance. However, the continuous presence of monocytes was detrimental to CART33 activity upon serial replating, as evidenced by suboptimal T cell expansion and loss of tumor control. To study the effects of normal myelopoiesis on CAR T cell activity in vivo, NSGS mice (NOD/SCID/IL2Rg null mice with human SCF, IL-3 and GM-CSF expression) were injected with human cord blood CD34+ cells to engraft human hematopoiesis. Mice were then injected with THP1 +19 tumor cells, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from the same cord blood product as the CD34+ cells. Consistent with our in vitro data, CART19 and CART33 showed better anti-tumor activity in the presence of human hematopoiesis. Notably, while the anti-tumor activity of CART19 and CART33 was equivalent in non-humanized mice, CART19 had better long-term activity in humanized mice, as mice treated with CART33 had late tumor relapses. To directly compare the effects of myeloid cells as bystanders versus targets, we lentivirally transduced cord blood CD34+ cells with human CD19 so that they would be targets for CART19. We then differentiated these cells into myeloid progenitors (MP) by culturing in IMDM with 20% FBS and the following cytokines: SCF (100ng/ml), FLT3L (50ng/ml), IL-3 (10ng/ml), and GM-CSF (10ng/ml). After 10 days of culture >95% of MP expressed CD33, and were selected for >95% CD19 expression (Fig A). Addition of non-transduced MP to CAR T cell and Ramos +33 co-culture enhanced CART19-mediated tumor clearance without affecting CART19 expansion. However, the presence of MP CD19+ had a detrimental effect on both tumor control and expansion of CART19, confirming that CAR T cells are less effective when targeting myeloid cells. In contrast, both MP and MP CD19+ had similar effects on CART33 that mirrored the negative effects of MP CD19+ on CART19 (Fig B). To summarize, we show that normal myeloid cells have beneficial effects on CAR T cell function, and targeting these same myeloid cells is detrimental to CAR T cell activity. These findings provide insight into the reason for suboptimal CAR T cell activity in AML, and show that independent of target antigen and CAR construct, the nature of the cells being targeted can change the effectiveness of this therapy.

Disparities in the Enrollment of Racialized, Ethnic Minority, and Older Adults in Randomized Trials of Acute Myeloid Leukemia: A Systematic Review

Introduction: There are significant racial and ethnic disparities in incidence and survival for patients diagnosed with hematologic malignancies. Understanding discrepancies in enrollment in randomized controlled trials based on race, ethnicity, and age is important to better understand access to care and clinical trial conduct. Objective: To systematically review the literature on enrollment of racialized, ethnic minority, and older adults in randomized controlled trials (RCTs) of acute myeloid leukemia (AML) and to provide enrollment estimates and compare these to population characteristics. Methods: MEDLINE was searched from inception through to June 2023. No restrictions based on language or publication date were used. Pairs of reviewers independently screened titles, abstracts, and full texts of records. Inclusion criteria were phase II and III RCTs of disease modifying therapy for AML in adults (≥18 years) reporting efficacy and safety outcomes. We excluded single arm trials, trials with unpublished results, conference abstracts, follow-up reports, subgroup/post-hoc/exploratory analyses, and supportive care trials, except those evaluating disease-modifying therapies and reporting clinical efficacy. A standardized form was pilot-tested and used to extract data related to trial characteristics. The proportion of trials reporting racial and ethnic subgroups (African American/Black, Asian, American Indian/Alaskan native, Native Hawaiian or other Pacific Islander, White, Hispanic), and age ≥65 were determined. For US trials, we calculated the enrollment incidence ratios (EIRs), the ratio of trial proportions of members of a racial and ethnic subgroup divided by US population-based incidence in the corresponding racial and ethnic subgroup using the Surveillance, Epidemiology, and End Results (SEER 20) database. We conducted a random-effects meta-analysis to pool EIRs. Results: After screening 7,759 titles and abstracts and 157 full texts, we included 90 studies, of which 14 (15%) were US trials. Overall, there were 21 (23.3%) trials that reported race or ethnicity and 26 (28.9%) that reported the enrollment proportion of ≥65 years. Of the trials with data on race or ethnicity, 15 (71.4%) had data on African American/Blacks, 21 (100%) Whites, 14 (66.7%) Asian or Pacific Islanders, 2 (9.52%) American Indian and Alaskan Native, 4 (19.0%) Hispanics. Of trials reporting on race, 176 (3.6%) of participants were African American/Black, 360 (7.2%) Asian or Pacific Islander, 5 (0.1%) American Indian and Alaskan Native, 3,914 (79.6%) White, and 39 (0.8%) Hispanic. Of the 14 US trials, 4 (28.6%) reported race and 4 (28.6%) reported enrolled proportion of older adults. Hispanic patients (EIR 0.20; 95%CI 0.07 to 0.59, I 2=86%), and Asian patients (EIR 0.24; 95%CI 0.07 to 0.86, I 2=77%) were significantly underrepresented while White patients (EIR 1.33, 95%CI 1.07 to 1.66, I 2=99%) were significantly overrepresented. Confidence intervals were wide for EIR of Black patients (EIR 0.96, 95%CI 0.42 to 2.2, I2=91%). Conclusion: Most trials did not report data on race and ethnicity or on enrollment proportion of participants ≥65 years. Only a small proportion of trial participants were from racial and ethnic minority groups. Hispanic and Asian patients were significantly underrepresented while White patients were overrepresented.

Targeting NUP214 Eradicates Leukemia Stem Cells By Inducing Ferroptosis

Acute myeloid leukemia (AML) arises from and are maintained by leukemia stem cell (LSCs), which are also critical causes for drug resistance and relapse of AML. The critical genes involved in LSC maintenance remain largely unknown. Here, we performed a pooled negative-selection screen with a customized small hairpin RNA (shRNA) library against 1191 known pan-essential genes of AML blast cells. We found that several Nucleoporins (NUPs) genes were essential for LSC survival, and NUP214 was highly expressed in LSCs. Knock-out of NUP214 completely eradicated LSCs in MLL-AF9 and HOXA9-MEIS1 AML mouse models, but also comprised HSC maintenance and function. Haploinsufficiency of NUP214 resulted in the increased death of LSCs (~4% to ~33%), reduced LSC numbers, and extended survival of AML mice. In contrast, the haploinsufficiency of NUP214 had a negligible effect on normal hematopoietic stem cells (HSCs) and hematopoiesis. Haploinsufficiency of NUP214 substantially induced lipid peroxidation and resulted in ferroptosis of LSCs, attributed to the remarkably increased transcription of lipoxygenase-15 (ALOX15) and heme oxygenase-1 (HMOX1), evidenced by the results of RNA-seq, proteome, and functional experiments. Immunofluorescence revealed that NUP214 was a mobile nucleoporin and largely located inside the nucleoplasm in LSCs, while NUP214 was mainly located in the nuclear periphery. Co-immunoprecipitation and Cleavage Under Targets and Tagmentation (CUT&Tag) showed that NUP214 directly interacted with transcriptional coactivator Sub1 in LSCs, binding the gene locus of HMOX1 and ALOX15, eventually inhibiting their transcription. Collectively, our findings reveal the essential roles of NUP214 in LSC maintenance and provide a valuable target for AML therapy.

A Systematic Review of Venetoclax and Azacitidine: Effectiveness and Safety for Newly Diagnosed and Relapsed Acute Myeloid Leukemia Patients

Introduction: Acute Myeloid Leukemia (AML) remains the most prevalent acute leukemia in adults, often associated with a bleak prognosis. Current treatment recommendations for elderly AML patients incorporate a combination of venetoclax (Ven) and azacitidine (AZA) following trials that underscored Ven's efficacy. This review seeks to analyze and summarize the literature concerning the effectiveness and safety of the Ven + AZA regimen for adult patients with newly diagnosed (ND) and relapsed/refractory (R/R) AML, and those unsuitable for intensive chemotherapy. Materials and Methods: Following PRISMA guidelines, a comprehensive electronic literature search was conducted across PubMed, Embase, Clinicaltrials.gov, Cochrane Library, and Web of Science from inception to 2023. MeSH terms and relevant keywords for (Leukemia, Myeloid, Acute) AND (Venetoclax) AND (Azacitidine) were used. From 327 initially identified articles, 12 studies were selected for inclusion, and citation searching further added 2 studies, summing up to 14. We analyzed data from a combined patient pool of 1256. Results: The review encompassed 13 non-randomized and 1 randomized clinical trials. The median patient age was 75.2 years. Primary outcomes comprised complete remission (CR) and complete remission with incomplete count recovery (CRi), yielding a pooled CR/CRi of 57.8%. Secondary outcomes included an overall response rate (ORR) of 61.1%, and a median overall survival (mOS) of 8.94 months with a median follow-up duration of 13.25 months (Table 1). Notably, high-grade adverse effects were predominantly hematological (anemia, neutropenia, thrombocytopenia, febrile neutropenia) while low-grade effects primarily involved gastrointestinal symptoms (nausea, vomiting, diarrhea, constipation, decreased appetite, etc). Conclusion: The current data suggest that the Ven + AZA regimen is both efficacious and well-tolerated for treating newly diagnosed and relapsed/refractory AML. Cytopenias across all three cell lines emerged as the most reported adverse effects of Ven + AZA therapy. Ongoing Phase III clinical trials are expected to enhance our understanding of this regimen's efficacy and safety profile, solidifying its role in standard AML therapy.

Safety, Pharmacodynamic, and Anti-Tumor Activity of SL-172154 As Monotherapy and in Combination with Azacitidine (AZA) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Patients (pts)

Background: CD47 blocking antibodies and SIRPα-Fc fusion proteins have demonstrated activity in combination with azacitidine (AZA) in previously untreated (UnTx) HR-MDS and AML pts. SL-172154 (SIRPα-Fc-CD40L), a hexameric, bi-functional fusion protein consisting of SIRPα domains linked to CD40L domains through an inert Fc linker demonstrated improved anti-tumor activity in comparison to naked CD47 blocking antibodies in pre-clinical studies [de Silva et al. Cancer Immunol Res 2020]. SL-172154, as a CD47 inhibitor, requires combination with AZA to enhance pro-phagocytic signals on leukemic stem cells/blasts thereby potentiating macrophage phagocytosis in AML/HR-MDS. Here we report the results from the Phase 1 Dose Escalation. cohorts for SL-172154 monotherapy [SL-mono] and for SL-172154 + AZA combination [SL-AZA] in pts with HR-MDS or AML. Methods: The objectives of the dose escalation cohorts include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic effects and efficacy per IWG 2006 for MDS and ELN 2017 for AML. SL-172154 was administered IV weekly of a 28-day cycle as monotherapy or with AZA 75 mg/m 2 for 7 days per cycle. mTPI-2 was used for dose escalation with a DLT threshold of 20%. Pts ≥ 18 years old with R/R HR-MDS or R/R AML were eligible for dose escalation cohorts. In addition, UnTx pts with TP53 mutant (TP53m)-MDS were eligible for SL-AZA cohorts. Results: As of May 25, 2023, 37 pts were enrolled (19 in SL-mono; 18 in SL-AZA cohorts): 14 women; median age 70 years [range 44-81]. 27 pts had R/R AML (16 de novo; 11 secondary), 5 had R/R MDS, and 5 were UnTx TP53m-MDS. 23 (85%) of 27 R/R AML pts had adverse genetic abnormalities per 2017 ELN classification, including 9 (33%) with TP53m or del 17. The median number of prior lines of AML therapy was 2 [range 1-4]; 25 (93%) had received venetoclax (VEN) and 23 (85%) had received a hypomethylating agent (HMA). All 5 pts with R/R MDS had previously received HMA and 2 pts were previously treated with VEN. SL-172154 was dose escalated as monotherapy or with AZA as shown in Table 1. Infusion-related reactions (IRRs) were the most common SL-172154-related treatment-emergent AEs (TEAEs) reported in 13 (68%) and 8 (44%) pts in SL-mono and SL-AZA cohorts, respectively. IRRs (51 events) were Grade (G) 1 or 2 except for 3 G3 events (1 each at 6 mg/kg SL-mono and SL-AZA; 1 at 3 mg/kg SL-mono). Other SL-172154-related TEAEs observed in ≥ 3 pts were AST increased (4; 21%), ALT increased (3; 16%) and nausea (3; 17%) in SL-mono cohorts, and nausea (3; 17%) in SL-AZA cohorts. All events of AST/ALT increased were transient. Only one DLT (G3 IRR in the 6 mg/kg SL-AZA cohort) was reported, which required a dose reduction to 3 mg/kg. As of July 10, 2023, in SL-mono cohorts, 1 pt with R/R AML post 7+3, FLAG and VEN/AZA achieved Morphologic Leukemia-Free State (blast reduction from 19% to <5%) after 1 cycle of 6 mg/kg SL-172154 and proceeded to allogeneic hematopoietic cell transplant (allo-HCT) after cycle 2. In 12 evaluable pts with R/R AML receiving SL-AZA, although no objective responses (OR) were observed, relative reduction in bone marrow (BM) blasts from baseline was reported in 2/5 pts at 1 mg SL-AZA (-50%, -75%) and 5/7 pts at 3 mg SL-AZA (ranging from -35% to -90%). One pt underwent an allo-HCT. In 4 evaluable pts with UnTx TP53m-MDS, the ORs were 1 complete remission (CR), 1 marrow CR, and 2 stable disease. 2 of them proceeded to allo-HCT. SL-172154 induced elevations in serum IL-12p40, IP-10, IL-8, IL-10, MIP3α and MCP1: greater response at 3 mg/kg compared to 1 mg/kg while similar between 3 and 6 mg/kg. In the BM, SL-172154 induced a dose-dependent increase in phagocytic cells (CD11b, HLA-DR, CD16, CD36 and CD64) within mature myeloid immune cell compartments (CD45highCD34-). Reduction in leukemic blasts (CD45lowCD34+) was associated with an increase in mature myeloid and phagocytic cell phenotypes (3 mg/kg SL-AZA > 1 mg/kg SL-AZA). Conclusions: SL-172154 was well tolerated up to 3 mg/kg as monotherapy and in combination with AZA. Preliminary efficacy signals were detected in UnTx TP53m-MDS and R/R AML. A response to SL-172154 monotherapy, dose-dependent increases in serum cytokines and accumulation of mature myeloid cells in BM suggest a potential role for CD40 stimulation. Based on the safety, preliminary efficacy and pharmacodynamic activity, 3 mg/kg SL-172154+ AZA is being evaluated in treatment naïve pts with TP53m AML and HR-MDS.

The Mevalonate Pathway Is a Therapeutic Target in TP53 Mutant Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) with mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a median overall survival of only six months due to chemotherapy resistance. To understand biologic differences in TP53 mutant (MT) AML, as compared to wildtype (WT) AML, we performed transcriptomic analysis on sorted patient samples. Gene set enrichment analysis demonstrated significant upregulation of the cholesterol biosynthesis or mevalonate pathway in TP53 MT AML. In TP53 MT solid tumor models, the mevalonate pathway plays a key role in tumorigenesis and metastasis. We hypothesized that the mevalonate pathway is essential for chemotherapy resistance of TP53 MT AML and represents a novel therapeutic target. Methods: We developed chemoresistant, isogenic TP53 MT AML cell lines using CRISPR/Cas9 technology in the MOLM14 AML cell line. We used primary TP53 MT AML patient samples to perform colony forming unit (CFU) assays, in vitro assays with a serum substitute and cytokines, and a patient derived xenograft (PDX) model of TP53 MT AML. Results: We first sought to determine if TP53 MT AML exhibits upregulation of mevalonate pathway activity in response to cytarabine (AraC), the backbone of AML therapy. We treated our isogenic TP53 MT AML cell lines for 24 hours (h) with AraC and assessed mevalonate pathway gene expression and metabolites using qRT-PCR and liquid chromatography high resolution mass spectrometry (LC-HRMS), respectively. Only TP53 MT cell lines respond to AraC with a significant upregulation of key mevalonate pathway genes and metabolites. We then determined if TP53 MT AML is sensitized to AraC by inhibition of the mevalonate pathway with a statin. We pretreated our isogenic cell lines with 24h rosuvastatin before 24h AraC and assessed cell viability with flow cytometry with AnnexinV/7AAD staining and XTT assays. We also performed CFU assays in TP53 MT AML patient samples treated with the two drugs alone or in combination and assessed CFUs after 14 days. These studies revealed a synergistic reduction in cell viability and CFUs in TP53 MT AML by rosuvastatin in combination with AraC. We next addressed the mechanism by which a statin sensitizes TP53 MT AML to AraC. Our group and others have demonstrated enhanced mitochondrial activity is associated with AML chemoresistance. We hypothesized that mevalonate pathway byproducts, known to be crucial for mitochondrial functions, contribute to the mitochondrial response to AraC. We treated TP53 MT AML cell lines as above with rosuvastatin and AraC and used seahorse technology to measure oxidative phosphorylation (OXPHOS). We also used electron microscopy, flow cytometry of mitochondrial protein, TOM20, and qRT-PCR for mitochondrial DNA content to quantify and characterize mitochondria. These data demonstrate that TP53 MT AML cell lines, compared to WT, exhibit a significant increase in OXPHOS after 24h that is due to an increase in total mitochondrial. These effects are abrogated by pretreatment with a statin. We also validated these findings in primary TP53 MT AML patient samples in vitro. Importantly, co-treatment of the isogenic TP53 MT AML cell lines with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. Overall, this data supports a model where TP53 MT AML cells dynamically upregulate the mevalonate pathway to regulate OXPHOS and avoid DNA damage-induced cell death (Figure 1). Finally, we studied a PDX model of TP53 MT AML. Engrafted mice were treated with AraC for five days alone or in combination with high dose rosuvastatin. Mice were harvested on day eight with assessment of leukemic burden by flow, OXPHOS by seahorse and mevalonate byproducts by LC-HRMS. TP53 MT AML leukemic burden was only significantly reduced by the combination therapy. Consistent with in vitro findings, mice sacrificed three days after completing AraC continue to demonstrate enhanced OXPHOS and increased mevalonate byproducts. This response is blunted by rosuvastatin. Conclusions: These results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of this pathway in combination with chemotherapy may improve clinical responses. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells in order to elucidate novel therapeutic approaches.

Synergistic Effect of CK2 Inhibitor CX-4945 with HDACi Chidamide on Cell Proliferation Arrest and Apoptosis in Acute Myeloid Leukemia

Background CX-4945 is an ATP-competitive inhibitor of CK2, which is currently being used in clinical trials to treat various cancer types. Chidamide (CHI), a novel histone deacetylase inhibitor (HDACi), is now involved in the salvage treatment of relapsed/refractory acute myeloid leukemia (AML) in a clinical trial. The anti-leukemia effects of either single drug have been reported by inducing cell apoptosis through suppressing multi-oncogenic pathways in human tumor cells. However, whether and how the combination of the two drugs exerts synergistic anti-tumor effects remains undetermined. This study is to investigate the synergistic therapeutic effects of CX-4945 in combination with CHI in AML. Methods Cell Counting Kit-8 (CCK-8) cell proliferation assay, Annexin V apoptosis assay, and Propidium Iodide cell cycle assay were performed in AML cells in the presence of CX-4945 only, CHI only, CX-4945 + CHI, and vehicle control for 48h. RNA-seq was performed in U937 cells treated with CX-4945 and CHI, respectively; the differentially expressed genes (DEGs) were identified by R studio, and pathway enrichment of the DEGs was analyzed with Metascape online tool. Data were expressed as the mean ± standard deviation. Ordinary one-way ANOVA statistical analysis and Log-rank test was employed to determine the significant difference between groups. A synergistic effect was determined by the combination indices (CIs) with CalcuSyn software. Results The 50% inhibiting concentration (IC50) of CX-4945 on the U937 and THP-1 cell proliferation is 7.435±0.066μmol/L and 6.787±0.153μmol/L. The IC50 of CHI on the U937 and THP-1 cell proliferation is 3.009±0.32μmol/L and 3.782±0.239μmol/L, respectively. The combination of various doses of CHI with either IC50 or 1/2 IC50 of CX-4945 significantly decreases the cell viability of the cells versus single-drug control and CalcuSyn analysis showed the high synergistic therapeutic effects of CHI with CX-4945 (Fig.1A). The combination of (4µM) CHI with (8µM) CX-4945 significantly induced the G0/G1 cell cycle arrest (p <0.001, Fig.1B) and elevated the % of apoptotic cells (p <0.0001, Fig.1C) in the AML cells compared to either single drug alone. Moreover, the combination of (1.6µM) CHI with (8µM) CX-4945 significantly increased the % of apoptotic cells in the primary blasts from the AML patient versus either single drug alone (p<0.0001, Fig.1D). To further understand the underlying mechanism of the synergy, RNA-seq analysis was performed in U937 cells treated with either CHI or CX-4945, respectively (Fig.2A). The DEGs are achieved upon either drug treatment, and the 76 up-regulated and 411 down-regulated overlapped DEGs were identified (Fig.2B). Pathways analysis showed that the overlapped DEGs are enriched in cell cycle progress, apoptosis, and multiple oncogenic signaling including cMyc and PI3K/AKT signaling pathways (Fig.2C). cMyc and PI3K/AKT-dependent signaling pathways are involved in myeloid cell proliferation. Also, our ChIP-seq data indicated obvious Ikaros binding peaks in the promoter region of cMyc and PIK3CD and they are the direct targets of Ikaros (data not shown). cMyc and PIK3CD are at the top of the overlapped DEGs, indicating the critical roles of the cMyc and PIK3CD underlying the mechanism of the combination in AML. Indeed, the mRNA level of cMyc and PIK3CD is significantly down-regulated upon the combination treatment versus single drug control (Fig.2D or data not shown). Consistently, the mRNA level of PIK3CD is significantly higher expressed in AML patients versus that in bone marrow samples of the healthy donors (p<0.05, Fig.2E) and the AML patients with high PIK3CD high expression have shorter OS than that with low expression (p <0.05, Fig.2F). These data support the hypothesis that the combination exerts the anti-tumor effect by suppression of cMyc and PIK3CD expression in AML. Conclusions The combination of CHI and CX-4945 has a synergistic effect on cell proliferation arrest, apoptosis, and cell cycle arrest by suppressing cMyc and PI3K signaling in AML cells. Our data provide experimental evidence for the combination as a potential option for AML therapy. More pre-clinical studies will be explored for future clinical trials.

Reconsidering the Significance of Blast Cut-Offs in Differentiating between MDS and AML Biology

Background The diagnosis of and distinction between acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS) have long been based on blast counts, with the presence of ≥20% myeloblasts in marrow or peripheral blood required to define AML in World Health Organization classification. Until 2001, blast counts greater than 30% had been used to diagnose AML by the original French-American-British classification system. However, the 2022 International Consensus Classification proposed a new combined category of MDS/AML with 10-19% blasts. Here we report the real-world treatment pattern and outcomes of AML and high-risk MDS patients at an academic institution in the United States, with specific attention to the blast burden at initial disease diagnosis to validate accuracy and limitation of this new proposed change. Methods This is a retrospective study conducted at UCLA Health encompassing all diagnoses of AML and MDS made at academic campus and community practices. We evaluated adult patients with a diagnosis with either AML or MDS from 1/1/2016-7/31/2022. For patients with more than one bone marrow biopsy on record, we recognized the date of diagnosis of MDS or AML with a blast burden ≥ 10% by immunohistochemistry or flow cytometry as the first bone marrow biopsy. In light of the changing diagnostic definitions, we analyzed patients by cohorts of MDS/AML with 10-19% blasts, AML low-blast with 20-29% blasts, and AML high-blast ≥ 30% blasts. Survival analysis by blast count at diagnosis was calculated by Kaplan-Meier estimates. A Multivariable Cox proportional hazard model was used to model survival outcomes by blast cohort, ELN risk 2022, response to first treatment, and receipt of allogeneic hematopoietic cell transplantation (HCT). Results There were 422 patients at UCLA Health with a diagnosis of AML or MDS included in our analysis with blast burden ≥ 10%. Patient demographics are shown in Table 1. Median age at diagnosis for MDS/AML, AML low-blast and AML high-blast cohorts is 67, 66 and 64 respectively (p=0.02). Thirty two (66.7%) patients in MDS/AML cohort received non-intensive first line treatment, whereas 202 (67.8%) AML high-blast patients received intensive first treatment. For AML low-blast patients, 37 (48.7%) received intensive treatment, and 29 (38.2%) received non-intensive treatment. More patients with AML (either low- or high-blast) underwent allogeneic HCT compared to MDS/AML patients. By Kaplain Meier analysis (KM) using blast counts as the sole predictor (Figure 1), no significant difference in OS when following patients for up to five years from diagnosis (p=0.0985) was seen. The p-value for two-way KM comparisons for MDS/AML vs. AML low-blast is 0.9682, and for AML low-blast vs. AML high-blast is 0.112. By multivariable analysis, ELN Risk 2022 (p<.0001), allogeneic HCT (p<.0001), and Complete Response to initial treatment (p<.0001) were predictive of OS, but blast percentages were not (p=0.56). Conclusion In our study, we found similar survival outcomes in high-risk MDS and AML patients regardless of initial blast counts at diagnosis. Though not statistically significant, MDS/AML and AML with low-blast burden appear to share more similar outcomes. This could be due to increased secondary AML seen in the low-blast cohort, and implies potentially different disease biology between high blast burden AML and AML with dysplastic changes. With this observation, we emphasize caution on simply using ICC 2022 to apply traditional AML therapy to the new MDS/AML category. Both WHO and ICC classification in 2022 reflects the increasing potential of molecular profiling to subtype these diseases, which will be the future direction. Rather than moving the blast count cutoff between AML and MDS, it is more critical to emphasize inclusion of high-risk MDS patients on all AML clinical trials and perhaps the converse for low blast burden AML with dysplastic changes, which might allow better understanding of response to therapy and disease biology.

Diagnosis of myelodysplastic syndromes after cytotoxic therapy for acute myeloid leukemia in the era of molecular research, difficulties in risk stratification and choice of therapy: The first domestic case report and literature review

Diagnosis of myelodysplastic syndromes after cytotoxic therapy for acute myeloid leukemia in the era of molecular research, difficulties in risk stratification and choice of therapy: The first domestic case report and literature review

Perspectives and challenges of small molecule inhibitor therapy for FLT3-mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease characterized overall by an aggressive clinical course. The underlying genetic abnormalities present in leukemic cells contribute significantly to the AML phenotype. Mutations in FMS-like tyrosine kinase 3 (FLT3) are one of the most common genetic abnormalities identified in AML, and the presence of these mutations strongly influences disease presentation and negatively impacts prognosis. Since mutations in FLT3 were identified in AML, they have been recognized as a valid therapeutic target resulting in decades of research to develop effective small molecule inhibitor treatment that could improve outcome for these patients. Despite the approval of several FLT3 inhibitors over the last couple of years, the treatment of patients with FLT3-mutated AML remains challenging and many questions still need to be addressed. This review will provide an up-to-date overview of our current understanding of FLT3-mutated AML and discuss what the current status is of the available FLT3 inhibitors for the day-to-day management of this aggressive disease.

LAIR-1 agonism as a therapy for acute myeloid leukemia.

Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.

Treatment of Acute Myeloid Leukemia in Older Adults.

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy predominantly affecting older adults. Despite the advancements in new therapies for AML, older and medically unfit patients continue to suffer from poor outcomes due to disease-related factors such as the mutational profile and patient-related factors such as comorbidities and performance status. In this review, we discuss a spectrum of therapeutic options for older patients with AML starting with a historical perspective and ending with therapies being investigated in clinical trials. We review the standard of care treatment options including combination venetoclax and hypomethylating agents, in addition to targeted therapies such as FLT3 and IDH inhibitors. Lastly, we shed light on challenges facing the care of older adults and their representation in clinical trials.

Survival outcomes in patients with acute myeloid leukaemia who received subsequent therapy for relapse in QUAZAR AML-001.

In the phase 3 QUAZAR AML-001 trial (NCT01757535) of patients with acute myeloid leukaemia (AML) in remission following intensive chemotherapy (IC) and ineligible for haematopoietic stem cell transplant (HSCT), oral azacitidine (Oral-AZA) maintenance significantly prolonged overall survival (OS) versus placebo. The impact of subsequent treatment following maintenance has not been evaluated. In this post hoc analysis, OS was estimated for patients who received subsequent AML therapy, and by regimen received (IC or lower-intensity therapy). First subsequent therapy (FST) was administered after treatment discontinuation in 134/238 Oral-AZA and 173/234 placebo patients. OS from randomization in patients who received FST after Oral-AZA versus placebo was 17.8 versus 12.9 months (HR: 0.82 [95% CI: 0.64-1.04], median follow-up: 56.7 months); OS from FST was similar between arms. Among patients who received injectable hypomethylating agents as FST, median OS was 8.2 versus 4.9 months in the Oral-AZA versus placebo groups (HR: 0.66 [95% CI: 0.41-1.06]). Forty-eight patients (16/238 Oral-AZA, 32/234 placebo) received HSCT following treatment discontinuation, including six Oral-AZA patients still in first remission; Oral-AZA OS benefit persisted when censoring these patients. Oral-AZA maintenance can prolong AML remission duration without negatively impacting survival outcomes after salvage therapies.

Combination therapy of acute myeloid leukemia by dual PI3K/mTOR inhibitor BEZ235 and TLR-7/8 agonist R848 in murine model

BackgroundDue to the high relapse rate and toxicity of the common therapies in patients with acute myeloid leukemia (AML), modifications in the treatment strategies are required. The present study was conducted to determine the effects of combinational therapy with a dual PI3K/mTOR inhibitor, BEZ235, and TLR7/8 agonist, R848, on murine AML model. MethodsBEZ235 and R848 were administered to AML leukemic mice in either a single or combination treatment. Frequency of T-CD4+, T-CD8+, MDSCs, NK, exhausted T cells and the degranulation levels was measured via flow cytometry. The cytotoxicity and proliferation levels were evaluated by MTT assay. Then, the expression of iNOS, arginase-1, PD-L1, Gal-9, PVR, IFN-γ, TNF-α, IL-4, IL-10, IL-12 and IL-17 was investigated by Real-Time PCR. Organomegaly, body weight and survival rate were also monitored. ResultsFollowing combinational therapy with BEZ235 and R848, increasing in the frequency of anti-tumor immune cells including T-CD4+ cells and M1 macroghages, and decreasing in pro-tumor immune cells including MDSCs, exhausted T-CD4+ and T-CD8+ cells and also M2 macrophages were observed. The functional defects of immune cells in term of proliferation, cytotoxicity, degranulation, and cytokines expression were improved in leukemic mice after treatment with BEZ235 and R848. Finally, organomegaly, body weight and survival analysis showed significant improvements after treatment with BEZ235 and R848. ConclusionTaken together, we indicated that the combinational therapy with BEZ235 and R848 could be considered as a potential and powerful therapeutic option for AML patients. Further clinical studies are required to expand our current findings.

CEBPA-Driven Expression of the Transcriptionally Inactive deltaTP73 Isoform Phenocopies TP53mutated Poor Risk and Drug-Resistant Acute Myeloid Leukemia

Mutations in TP53 ( TP53mut) are present in roughly 10% of acute myeloid leukemia (AML) patients and represent a unique subgroup of patients with very poor prognosis. While the exact mechanisms of TP53mut-driven leukemogenesis remain elusive, transcriptional changes associated with suppression of the TP53 signaling pathway are considered to confer a dismal prognosis on TP53mut patients. Using molecular profiling of a large cohort of AML patients (n=823) we identify a new poor prognostic subgroup in AML that is TP53 WT but shares strong similarities with TP53mut patients. Patients included in this new group present increased expression of the oncogenic TP73 gene isoform ΔNp73,which lacks the transactivation domain but still binds to chromatin. We find that the transcriptional and metabolic program of ΔNp73high AMLs strongly resembles that of TP53mut AML, being enriched for stemness signatures like “17LSC” and “LSC UP”. In line with TP53mut AML, ΔNp73high/ TP53WT frequently co-occurs with U2AF1, SRSF2, TET2, and RUNX1 mutations . Lentiviral overexpression (OE) of ΔNp73 in healthy CD34 + cells increased cell proliferation and stemness. ΔNp73-OE in TP53WT AML models, imposed drug resistance against several standard-of-care cytotoxic therapies in AML (e.g., AraC, venetoclax (VEN) ± azacytidine (AZA), and FLT3 inhibitors), while drug resistance was not further enhanced upon ΔNp73-OE in TP53mut AML models. These results were validated using ex vivo treated primary AML samples (n=80), whereby ΔNp73 high samples were more resistant to several cytotoxic therapies, including VEN+AZA . Lentiviral ΔNp73-OE in primary APL samples (n=10) allowed efficient engraftment in vivo using xenograft mouse models with colonization of secondary organs and splenomegaly, which was not observed in the empty vector controls. To unravel mechanisms via which ΔNp73controls oncogenic transformation we performed RNA-seq and ChIP-seq to identify direct transcriptional targets of TP53, ΔNp73, and the transcriptionally active isoform TAp73. These studies revealed that ΔNp73 and TP53 compete for the same target genes, whereby ΔNp73-OE resulted in complete loss of TP53 binding and strong downregulation of these loci, including downregulation of known TP53 target genes CDKN1A, BBC3, and GAS6, but also of TP53 itself. Gene set enrichment analyses of molecular programs in ΔNp73-OE cells revealed that these were associated with the terms “LSC UP”, “CEBPA targets”, and “SIGNAL TRANSDUCTION BY TP53”, indicating that ΔNp73 phenocopies TP53mut AML. Chromatin accessibility data on AML patients revealed a differentially accessible region in intron 2 (+24 Kb after the transcription start site, referred to as peak B) of the TP73 gene, with unknown regulatory functions on TP73 expression in AML. To access the potential enhancer functions of this region driving ΔNp73 expression we generated CRISPR/Cas9-KO MOLM13 cells lacking the peak B region (MOLM13-KO). MOLM13-KO cells exhibited reduced ΔNp73 expression, and the ability to up-regulate ΔNp73 expression upon high dose AraC-induced stress was abrogated. Since CEBPA was a transcription factor predicted to bind to Peak B, we investigated whether CEBPA could regulate the expression of the different TP73 isoforms. CEBPA-KD in AML cells resulted in decreased ΔNp73 levels and restored the TAp73 expression, suggesting that CEBPA controls the balance of the distinct TP73 isoforms. Pharmacological inhibition of CEBPA using the FDA-approved drug Guanfacine (GFC), diminished ΔNp73 levels and restored drug sensitivity against several cytotoxic agents in ΔNp73-OE AML cells. Moreover, the combination of GFC+VEN displayed strong synergistic effects in a cohort of ex vivo treated AML blasts (n=20), including patients with TP53mut, which are often resistant to VEN. Here, we describe a new AML patient subgroup that is TP53WT but behaves similarly to TP53mut AML due to the expression of the ΔNp73 isoform that impairs TP53 signaling. This patient subgroup displays a very poor prognosis and is resistant to standard-of-care drugs used for AML patients. We identify a novel enhancer region that drives the expression of oncogenic TP73 isoforms, which is controlled by CEBPA, and inhibition of CEBPA by GFC enhances sensitivity to cytotoxic therapies in TP53mut-like AMLs. Our insightsfurther refine patient stratification and provide alternatives for treatment for the most dismal group of AML patients.

Outcomes of Patients Receiving Intensive Therapy for Acute Myeloid Leukemia Relapsed after/Refractory to Frontline Less-Intensive Therapy

Background: Hypomethylating agent (HMA) and venetoclax (Ven) frontline therapy for acute myeloid leukemia (AML) is astandard of care for patients (pts) deemed to be poor candidates for intensive induction due to age or fitness. However, given its attractive risk-benefit ratio, there is an increasing interest to extend the preference for HMA + Ven to other pts, including those who are fit for intensive therapy but with adverse risk disease. Unfortunately, most pts treated in this fashion will require future therapies for relapsed/refractory (R/R) disease and intensive therapy is given consideration mostly for pts with a path to allogeneic hematopoietic stem cell transplantation (alloHCT). Little is known about the outcomes of pts who receive intensive induction therapy after use of a frontline less-intensive regimen in the modern era. In this multi-center study, we seek to describe the experience with such a patient population and identify predictors of differential clinical outcomes. Methods: We conducted a multi-center, retrospective study of pts with AML treated with frontline less-intensive chemotherapy, followed by intensive induction chemotherapy for R/R disease. We first described the baseline characteristics of this cohort. Nominal variables were summarized with frequency and percentage while continuous variables were summarized with median and interquartile range (IQR). Overall response rate (ORR) was defined as complete remission (CR), complete remission with incomplete count recovery, or morphologic leukemia free state. Differences in ORR were analyzed with Fisher's exact test for nominal variables and Wilcoxon rank-sum test was used for continuous variables. Kaplan-Meier analysis was used to determine median overall survival (OS) after induction chemotherapy. Log-rank testing and Cox proportional hazard models were used to determine survival differences for nominal and continuous variables, respectively. Statistical significance was determined as p-value <0.05. Results: A total of 23 pts were included in this study. Median age at diagnosis was 64 years (IQR: 62-71). The majority of pts were male (n=12, 52%), white (n=20, 87%), and had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 (n=18, 82%). The majority of pts (61%) at time of frontline therapy had AML arising out of MDS. As expected, the majority of pts had adverse risk ELN cytogenetics (n=16, 70%) with an enrichment for disease harboring TP53 mutation (n=6, 27%) and complex karyotype (n=9, 39%). Fifteen pts (65%) had frontline therapy with azacitidine backbone, 6 pts (26%) with decitabine; some pts received both before intensive therapy. The median number of cycles of frontline therapy was 2 (IQR: 2-5). Median WBC at time of R/R therapy was 1.9 x 10 9/L (IQR: 0.8-6.45), while hemoglobin was 8.4 g/dL (IQR: 7.5-9.45), platelets were 41 x 10 9/L (IQR: 19.5-116.5), and marrow blasts were 25% (IQR: 16.5-30%). The plurality of pts received CPX-351 as intensive R/R therapy (n=9, 39%). Other regimens included 7+3 (n=2, 9%), FLAG-Ida + Ven (n=2, 9%), HiDAC + Mitoxantrone +/- Ven (n=4, 17%) ( Table 1). ORR after intensive R/R therapy was 27% (n=6), with 23% achieving CR. CPX-351 after HMA + Ven was specifically associated with an ORR of 33% (CR rate 22%). Median OS after initiation was 5.8 months. Thirty- and 60-day mortality was 17% and 26%, respectively. Five patients (23%) proceeded to alloHCT ( Table 1). Univariate differences in response rate and median survival were analyzed for each of the aforementioned variables as illustrated in Table 1. While none of the covariates reached statistical significance for impacting ORR or OS, ECOG PS trended towards significantly impacting OS ( p=0.074) and the median OS among patients proceeding to alloHCT was numerically doubled (8.0 vs. 4.0 months, p=0.11) ( Figure 1). Conclusions: Intensive therapy for R/R AML after prior frontline less-intensive therapy was associated with an ORR rate of 27% and median OS of 5.8 months in our cohort. This approach may be an appropriate strategy for a subset of pts, particularly those eligible for alloHCT. The relatively small and heterogeneous nature of this cohort limits strong conclusions regarding efficacy. As the use of frontline HMA + Ven continues to expand, further studies with larger sample size are needed to determine which pts would and would not benefit from intensive induction therapy in the R/R setting.

COX4I1 Controls Mitochondrial Electron Transport Chain Complex IV Assembly and Leukemia Progression in Acute Myeloid Leukemia

Aberrant cellular signaling plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Identifying key signaling components and their dependencies is crucial for advancing AML therapy. Here, we conducted a focused CRISPR library screen targeting 427 cell signaling-related genes with 3,416 sgRNAs (8 sgRNAs per gene) in the Cas9-expressing Molm13 AML cells. In addition to the known leukemia essential genes (MYC, RPS6, CCND3, GAB2, and AURKB), we discovered COX4I1 (Cytochrome c oxidase subunit 4 isoform 1) as a novel essential gene for AML maintenance (Fig. 1). CRISPR-knockout of COX4I1 in AML cells led to inhibited cell proliferation, reduced colony formation, and attenuated leukemia progression in vivo. Transcriptomic profiling revealed elevated mitochondrial stress upon COX4I1 knockout, accompanied by dysregulated mitochondrial cristae structure observed through transmission electron microscopy. Functional assays demonstrated impaired mitochondrial respiration (oxygen consumption rate, i.e., OCR, measured by Seahorse XF) and depleted cellular ATP (measured by metabolomic mass spectrometry) upon COX4I1 knockout in AML cells, highlighting the pivotal role of COX4I1 in mitochondrial homeostasis and energy metabolism. COX4I1 is a nuclear-encoded mitochondrial protein located on the mitochondrial inner membrane. Using a high-density CRISPR gene tiling scan (Fig. 2; targeting density ~2.1 aa per sgRNA), we identified two critical COX4I1 peptide regions (P1: K67 - L73 and P2: E136 - K169) required for leukemia cell proliferation and mitochondrial respiration. Mechanistically, we found that expression of COX4I1 is essential for the assembly of mitochondrial complex IV, a crucial component of the electron transport chain involved in establishing the proton electrochemical potential across the mitochondrial inner membrane. Structural analysis revealed that facing the mitochondrial matrix, COX4I1's P1 region establishes the interface between COX4I1 and COX5A, which is necessary for the formation of the COX1 module. Conversely, COX4I1's P2 region (C-terminal 34 residues) is dispensable to the COX1 module formation; nonetheless, the P2 region co-localizes with the intermembrane space domains of the COX2/COX3 modules and is required for the assembling of an intact complex IV. Finally, we found that knockout of COX4I1 induced mitochondrial stress further triggers cellular ferroptosis in AML cells, which is accompanied by an imbalanced cellular redox index (reduced GSH/GSSG ratio) and elevated lipid peroxidation (increased malondialdehyde). These notions led us to discover the critical role of COX4I1 as an opportunity for targeted AML therapy, especially when combined with mitochondria-targeting therapeutics such as Venetoclax (i.e., ABT-199). In conclusion, our study highlights COX4I1 as a novel vulnerability in AML, controlling mitochondrial complex IV assembly and ATP biosynthesis. This finding holds promise for improving AML therapy and may be extended to identify therapeutically relevant signaling components in other hematopoietic malignancies.