Subset Of Acute Myeloid Leukemia Research Articles

Molecular measurable residual disease monitoring and transplant indications in NPM1 mutated acute myeloid leukemia.

NPM1 mutated acute myeloid leukemia (AML) comprises roughly 30% of all AML cases and is mainly classified as favorable or intermediate-risk according to the European Leukemia Net stratification. Some patients, however, either have a poor response to initial intensive chemotherapy or ultimately relapse. NPM1 mutations are common, generally stable at early relapse and AML specific, features which make them ideal targets for measurable residual disease (MRD) monitoring. MRD monitoring via molecular analysis during the course of treatment can inform the role of allogeneic stem cell transplantation (HCT) in first remission in patients with NPM1 mutated AML with high-risk co-occurring mutations, particularly FLT3-ITD, and in favorable risk patients who do not achieve defined molecular milestones. In this review, we evaluate the prognostic role of MRD monitoring in NPM1 mutated AML and its use as a predictive biomarker to refine risk stratification and inform decision making regarding treatment. We explore the impact of pre-HCT MRD positivity on post-HCT outcomes in this AML subset, and how HCT-related factors such as conditioning intensity may influence this risk.

Expanding the therapeutic horizon in acute myeloid leukemia: The role of phosphoinositide-3-kinase-γ inhibition

Abstract Acute myeloid leukemia (AML) remains a formidable challenge due to its high relapse rate and limited therapeutic options. This study investigates the role of phosphoinositide-3-kinase-γ (PI3Kγ) in AML and explores the therapeutic potential of the PI3Kγ inhibitor, eganelisib, in preclinical models. Using genome-wide CRISPR interference screening, we identified PI3Kγ as a critical survival pathway in certain AML subsets. We demonstrated that inhibition of PI3Kγ with eganelisib disrupts oxidative phosphorylation, thereby impeding AML cell survival and proliferation. Notably, the combination of eganelisib with cytarabine showed enhanced efficacy, suggesting a synergistic interaction that improves treatment outcomes. These findings indicate that PI3Kγ is a viable therapeutic target in AML and that PI3Kγ inhibitors could be pivotal in overcoming treatment resistance and reducing relapse.

PTPN11 Mutation Clonal Hierarchy in Acute Myeloid Leukemia.

Mutations in protein tyrosine phosphatase non-receptor type 11 ( PTPN11 ) have been considered late acquired mutations in acute myeloid leukemia (AML) development. To interrogate the ontogeny of PTPN11 mutations, we utilized single-cell DNA sequencing and identified that PTPN11 mutations can occur as initiating events in some AML patients when accompanied by strong oncogenic drivers, commonly NPM1 mutations. The co-driver role of PTPN11 mutations was confirmed in a novel murine model that exhibits an AML phenotype with early expansion of a diverse set of variably differentiated myeloid cells that engrafted into immunodeficient and immunocompetent mice. This immune diversity was reconstituted from early precursor cells when engrafted into immunodeficient mice. Moreover, immune diversity was also observed in the blast component of patient samples with NPM1 and PTPN11 mutations, providing novel antigen targets for immune based approaches in this subset of AML that is resistant to multiple targeted therapies.

Pathogenicity role of human herpesvirus-8 in patients with acute myeloid leukemia

Abstract: BACKGROUND: Certain hematologic cancers, including Kaposi’s sarcoma (KS), have been associated with the pathogenicity of human herpesvirus-8 (HHV-8). HHV-8’s involvement in acute leukemia patients is yet unclear, nevertheless. The diagnosis, categorization, and course of treatment for acute myelogenous leukemia, an aggressive heterogeneous hematologic malignancy, have changed dramatically in recent years. OBJECTIVE: This study aims to investigate the pathogenicity role of HHV-8 in patients with acute myeloid leukemia (AML) of a group of the Iraqi population. MATERIALS AND METHODS: Case–control research has been carried out on 75 fresh blood samples recruited from the Mirjan Teaching Hospital in Al-Hilla City. The studied blood samples were obtained from patients with AML enrolled in this study, whereas control groups in the current study included 75 fresh whole blood. The specimens were collected during the period from June 2023 to February 2024. Conventional polymerase chain reaction (PCR) was used to identify HHV-8. RESULTS: The results show that the mean age of the patients with AML (48.5 ± 10.23 years) was more than that of the apparently healthy control (46.26 ± 11.21 years). There was a nonsignificant difference between patients with AML and the control group. In addition, the male in this study group constituted 56% (42/75), whereas 44% (33/75) were female. Furthermore, the positive of viral genome extraction was found in 41.3% (31 out of 75 of the specimens with viral genome), whereas 59.7% (44/75) specimens did not contain viral genome. The PCR results showed that in the AML patient group, the rate of HHV-8 infection was 35.4% (11 out of 31 cases). CONCLUSION: Considering the relatively small numbers included in our results, the positive results lead to the idea that HHV-8 works as a cofactor in the tumor biology of the AML subset under consideration and may have contributed to its development.

A prospective study of all-trans retinoic acid plus venetoclax and azacitidine in newly diagnosed acute myeloid leukemia.

6545 Background: Over the decade, anthracycline and cytarabine-based intensive chemotherapy remains the standard of care for acute myeloid leukemia (AML) but is hampered by serious toxicities like myelosuppression, increased transfusion requirement and disappointing clinical outcomes among the so-called poor-prognosis AML subsets. BCL-2 inhibitor plus azacitidine (AZA) has become a preferred treatment for AML, but the remission rate and overall survival (OS) are suboptimal. Apart from hematologic toxicities, 70.0% of patients were RBC transfusion dependent and 58.6% were platelet transfusion dependent. At present, all-trans retinoic acid (ATRA) is used as a differentiation drug, and there is no combination regimen with BCL-2 inhibitor. This study aimed to assess the efficacy and safety of ATRA plus venetoclax and AZA for newly diagnosed AML. Methods: This study enrolled 35 patients (≥18 years) with newly diagnosed AML between 2022 and 2023. For induction, patients received AZA (75 mg/m²) from Day 1 to 7, venetoclax at a target dose of 400 mg/d from Day 2 to 10, and ATRA (45 mg/m²/d) from Day 12 to 28 of each 28-day cycle for up to 2 cycles or until disease progression. Patients who achieved complete remission (CR) after two cycles of induction therapy received bone marrow transplantation or consolidation at their own discretion. For consolidation, patients received ATRA (45 mg/m²/d) from Day 1 to 21 and AZA (75 mg/m²) from Day 1 to 7 of each 28-day cycle for up to 4 cycles or until disease progression. For maintenance, patients received ATRA (45 mg/m²/d) for 21 days per cycle plus AZA (75 mg/m²). Maintenance therapy was given every 3 months until disease progression and ATRA was provided during intermissions. The primary endpoint was the composite CR rate (CR and CR with incomplete hematologic recovery) at the end of cycle 1 of inductive therapy. Results: As of now, the composite CR rate reached 74% (26/35) and the objective response rate reached 91% (32/35). The composite CR was 73% (11/15) in the adverse risk group and 75% (15/20) in the favorable-intermediate risk group at the end of cycle 1. Thirty-three patients entered cycle 2. The cumulative composite CR rate reached 91%(30/33) after 2 cycles of induction therapy. The median event-free survival was 390 days, and the median OS was 573 days. Platelet transfusion independence occurred in 17% (6/35) patients in cycle 1 and 73% (24/33) in cycle 2. Furthermore, 20% (7/35) and 73% (24/33) patients were RBC transfusion independent in cycle 1 and 2, respectively. Any-grade infections occurred in 74% (26/35) patients, including 12 before admission. Conclusions: The treatment regimen achieved a high composite CR rate and was well tolerated, with reduced hematologic toxicities in cycle 2 of induction therapy. Both adverse risk and favorable-intermediate risk patients benefited from the regimen, which holds promise for treating AML in adult patients. Clinical trial information: NCT05654194 .

The RTK-RAS signaling pathway is enriched in patients with rare acute myeloid leukemia harboring t(16;21)(p11;q22)/FUS::ERG.

Acute myeloid leukemia (AML) with t(16;21)(p11;q22)/FUS::ERG is a rare AML subtype associated with poor prognosis. However, its clinical and molecular features remain poorly defined. We determined the clinicopathological, genomic, and transcriptomic characteristics and outcomes of patients with AML harboring FUS::ERG at our center. Thirty-six AML patients harboring FUS::ERG were identified, with an incidence rate of 0.3%. These patients were characterized by high lactate dehydrogenase levels (median: 838.5 U/L), elevated bone marrow blast counts (median: 71.5%), and a CD56-positive immunophenotype (94.3%). Notably, we found that RTK-RAS GTPase (RAS) pathway genes, including NRAS (33%) and PTPN11 (24%), were frequently mutated in this subtype. Transcriptome analysis revealed enrichment of the phosphatidylinositol-3-kinase-Akt (PI3K-Akt), mitogen-activated protein kinase (MAPK), and RAS signaling pathways and upregulation of BCL2, the target of venetoclax, in FUS::ERG AML compared to RUNX1::RUNX1T1 AML, a more common AML subtype with good prognosis. The median event-free survival in patients with FUS::ERG AML was 11.9 (95% confidence interval [CI]: 9.0-not available [NA]) months and the median overall survival was 18.2 (95% CI: 12.4-NA) months. Allogeneic hematopoietic stem cell transplantation failed to improve outcomes. Overall, the high incidence of RTK-RAS pathway mutations and high expression of BCL2 may indicate promising therapeutic targets in this high-risk AML subset.

Metabolic reprogramming regulated by TRAF6 contributes to the leukemia progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.

Abstract 7161: Preclinical pharmacology modeling (pharmacokinetics/efficacy) of narazaciclib (HX301 or ON123300) to predict its clinical effective dose

Abstract Background: Acute myeloid leukemia (AML) is an aggressive leukemia of myeloid lineage. CSF1R (colony-stimulating factor 1 receptor) regulates the proliferation, differentiation and polarization of cells of myeloid lineages, e.g. monocytes/macrophages. CSF1R expression is aberrantly upregulated in many AML, leading to a hypothesis that CSF1R is a putative leukemogenic driver in a subset of AML and thus could be a potential target for treating AML. Previous studies demonstrated that Narazaciclib (HX301 or ON123300), an investigational kinase inhibitor (TKi), exhibited high potency activity against CSF1R (IC50 0.285 nM) and inhibition of macrophage proliferation and AML tumor growth both in vitro/in vivo, implicating its potential use as an AML therapeutic1 (An et al., 2023 AACR Annual Meeting). In addition, narazaciclib has recently completed the dose escalation phase 1 study in advanced solid tumors. However, the therapeutic window of narazaciclib as a CSF1Ri for treating AML remains to be determined. The present study attempted to utilize both preclinical PK and in vivo efficacy models to predict the effective clinical dose of narazaciclib for guiding the future AML clinical trials. Methods: A single dose of narazaciclib at various of dose levels was orally administrated in NOD/Scid mice, and plasma samples were collected at designated timepoints. The concentrations of narazaciclib in plasma were analyzed by LC-MS. In a Ba/F3-ETV6-CSF1R in vivo xenograft tumor model in Nod/Scid mice, narazaciclib at various dose levels was orally administrated daily. Tumor volumes were measured twice weekly. Results: The mouse PK study showed that narazaciclib displayed overall similar absorption pattern as seen in unpublished human clinical data. Narazaciclib was absorbed rapidly after oral dosing with a mean Tmax ranging from 1 to 2 hours in mouse. However, narazaciclib was cleared faster in mouse, with a mean T1/2 ranging from 1.9 to 2.8 hours, than in human. In addition, the exposures of narazaciclib, as measured by Cmax and AUC in plasma, increased in a relatively dose-proportional manner, as seen in human patients. In the Ba/F3-ETV6-CSF1R xenograft model where the tumor cell growth is solely dependent on the CSF1R kinase activity, narazaciclib at 10 mg/kg did not induce any anti-tumor effects with a TGI of -14.61%. In contrast, narazaciclib at 50 mg/kg and 100 mg/kg exerted significant anti-tumor effects with a TGI of 85.49% and 94.55%, respectively. Based on the mouse PK results, narazaciclib at 50 mg/kg in mouse is estimated to have an AUC of approximate 2000 h*ng/mL, which is equivalent to the dose of 160 - 200 mg/d in human patients. Therefore, 160 mg dose level is predicted to be an effective dose for narazaciclib in AML patients. Conclusions: Narazaciclib at 160 mg/d is predicted to be an effective dose in AML patients. Citation Format: Tao Yang, Hang Ke, Jinping Liu, Kefeng Gong, Henry Qixiang Li. Preclinical pharmacology modeling (pharmacokinetics/efficacy) of narazaciclib (HX301 or ON123300) to predict its clinical effective dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7161.

Abstract 5795: Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems

Abstract Phenotypic plasticity, or the ability of cancer cells to switch between cell states, has recently been recognized as a key hallmark of cancer. Cancer cells can hijack normal developmental pathways, preventing terminal differentiation or causing de-differentiation or transdifferentiation to a more proliferative, plastic cell state similar to those found in early development. Using our proprietary AI/ML platform AURIGIN, we created a high-resolution atlas to define the drivers of normal cell states during human development. After mapping primary human tumors to the atlas, we identified the histone acetyltransferase KAT2A as a key driver of tumor cell plasticity in a subset of acute myeloid leukemias (AML), small cell lung cancer (SCLC) and neuroendocrine prostate cancer (NEPC). Targeting KAT2A was genetically validated in MOLM-13 (AML) cells, where knockdown resulted in significant cell growth inhibition and induction of cell state changes consistent with a more mature differentiation phenotype. Similarly, an early tool heterobifunctional degrader of KAT2A and KAT2B (AUR101) resulted in potent growth inhibition and granulocytic differentiation in MOLM-13 cells and in 5/12 primary AML patient samples grown ex vivo. Treatment of NCI-H1048 SCLC cells with AUR101 potently degraded KAT2A/B, induced a significant shift from a dedifferentiated cell state to a more differentiated epithelial cell state and potently inhibited cell growth both in vitro and in vivo. The strong biological validation of KAT2A/B in SCLC and AML tumors, led us to develop AUR1545, a novel sub-nanomolar degrader of KAT2A and KAT2B. AUR1545, with its improved drug like properties, was tested across representative models of AML, SCLC and NEPC. In vitro, treatment with AUR1545 inhibited proliferation and promoted profound cell state changes in MOLM-13 (AML) cells. In NCI-H1048 (SCLC), LASPC-01 (NEPC) cell lines as well as a primary NEPC organoid model, AUR1545 demonstrated potent antiproliferative effects. In vivo, AUR1545 significantly inhibited tumor growth in an NCI-H1048 xenograft model. Taken together, these data genetically and pharmacologically validate KAT2A and KAT2B as key drivers of cell state plasticity in SCLC, NEPC and AML tumors and point to their utility as novel targets in these aggressive, metastatic and drug resistant cancers. Citation Format: Kimberly S. Straley, James Neef, Ileana Antony-Debre, Maulasri Bhatta, Sambad Sharma, Sara Sinicropi-Yao, Joe DeBartolo, Andrew McRiner, Betty Chan, Henry Wilson, Christina S. Lee, Zied Boudhraa, Mohamed El Ezzy, Umar Sharif, Mark Bittinger, Laura Antipov, Thomas G. Graeber, Stephane de Botton, Katharine E. Yen, David S. Millan. Potent and selective degradation of KAT2A and KAT2B induces profound cell state changes and inhibits growth of AML, SCLC and NEPC model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5795.

Abstract 5589: CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine

Abstract CBFB::MYH11 acute myeloid leukemia (AML) is a subset of Core Binding Factor AML initiated by an oncofusion in which Core Binding Factor Beta (CBFB) is fused to the C-terminus of smooth muscle myosin heavy chain 11 (MYH11). CBFB::MYH11 AML has a high cure rate with conventional anthracycline/cytarabine induction and high-dose cytarabine consolidation in patients under the age of 60, and remains chemosensitive after multiple relapses; the mechanisms underlying chemosensitivity are unknown. TP53 is a key regulator of DNA damage response and apoptosis, and TP53-mutated AML is often chemoresistant. Large sequencing studies have revealed only rare co-occurrence of TP53 mutations with CBFB::MYH11. We initially used CBFB::MYH11 as a negative control for an unrelated study of TP53 function and unexpectedly found striking synergy. While expression of CBFB::MYH11 in WT murine hematopoietic stem and progenitor cells (HSPCs) induced a low-penetrance (~20%), long latency (~6 months) myelomonocytic AML, expression of CBFB::MYH11 in Trp53-/- HSPCs induced AML with 100% penetrance and a median latency of 8 weeks. CBFB::MYH11-induced AMLs arising in Trp53-/- cells were similar by morphology and flow cytometry phenotype. We validated these findings in a second model, using a conditional Trp53flox/flox x Vav1-Cre model in which Trp53 is inactivated in HSPCs, with nearly identical results. Trp53-/- cells transduced with Empty Vector did not develop AML, but did develop T cell lymphoma with a median latency of 16 weeks, similar to prior studies. Trp53 deficiency also cooperated with RUNX1::RUNX1T1, the other Core Binding Factor oncofusion, to induce a high-penetrance (100%) and short latency AML (median latency 10 weeks); consistent with prior data, RUNX1::RUNX1T1 expression alone in Trp53 WT cells was insufficient to induce AML. To determine whether Trp53 influences the chemosensitivity of CBFB::MYH11 expressing cells, we performed cytarabine dose-response experiments. While CBFB::MYH11-transduced WT HSPCs were very sensitive to cytarabine (relative to EV), sensitivity was reduced in CBFB::MYH11-transduced Trp53-/- cells, suggesting that CBFB::MYH11-induced chemosensitivity is, at least in part, mediated by the TP53 pathway. To determine whether CBFB::MYH11 expression results in upregulation of key TP53 pathway genes, we transduced WT HSPCs with EV or CBFB::MYH11, transplanted cells into WT mice, harvested bone marrow at 3 months, and performed RNA-Seq. Key TP53 pathway genes were upregulated, including Bbc3/Puma, Bax, Cdkn1a/p21, and Trp53 (all 2.6-4.6-fold, p≤0.05). These data suggest that the chemosensitivity and the favorable prognosis of CBFB::MYH11 AML may be due to the direct activation of the TP53 pathway by CBFB::MYH11. Similar studies are underway with RUNX1::RUNX1T1-transduced cells to determine whether TP53 is relevant for the chemosensitivity of that CBF AML as well. Citation Format: Ryan B. Day, Julia A. Hickman, Casey D. Katerndahl, Sai Mukund Ramakrishnan, Christopher A. Miller, Timothy J. Ley. CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5589.

Abstract 3904: Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia

Abstract Introduction: Acute Myeloid Leukemia (AML) is a heterogenous hematological malignancy with a complex clonal architecture that requires in-depth immunophenotyping for disease characterization. Conventional AML ex vivo models for preclinical drug screening lack functional readouts that provide single cell level data to identify drug activity in defined AML subsets with unique immune signatures. Here, we present a novel platform that links Multiparameter Flow cytometry with high-throughput EX vivo drug screening (MFLEX) in AML patient-derived cell models to simultaneously detect phenotypic changes and predict cell-subset specific drug responses in AML. Method: We developed a 21-color high-dimensional immune profiling panel that captures AML heterogeneity by delineating primitive and mature AML blasts along with normal hematopoietic lineages. The panel also includes markers for functional measurement of apoptosis, proliferation, differentiation, and expression of BCL2 family proteins in lymphoid and myeloid cell subsets. Ex vivo culture conditions were optimized using physiologically relevant stroma-free cytokine-rich media that supports primary AML cells with minimal phenotypic shift, allowing for high throughput functional drug sensitivity testing. Timepoints and dosing conditions for AML standard of care (SoC) agents were optimized to support combination screens. Multi-parameter datasets were analysed for functional differences and visualized using dimension reduction methods. Results: We tested MFLEX on clinically annotated AML samples to understand sensitivity profiles to venetoclax and combination partners: 5-azacytidine, cytarabine, fludarabine and gilteritinib. We identified distinct drug sensitivity profiles for venetoclax in AML patients with varying leukemic differentiation states where M0/M1 subtypes were more sensitive to therapy than M4/M5. We also observed cell lineage specific sensitivities, with myeloid blast being more sensitive to venetoclax compared to autologous CD4+ and CD8+ T-cells and this observed difference was correlated with higher expression of BCL2. Our platform demonstrated that patients who previously failed venetoclax therapy could benefit from a combination with 5-azacytidine or fludarabine and this was patient-specific. We further evaluated MFLEX’s predictive value for patient clinical response by systematically comparing venetoclax sensitivity in patient-derived xenograft models in vivo vs ex vivo and found a high correlation between drug responses. Conclusion: We have established a unique, immune drug screening platform, MFLEX, that has high translational value and can be implemented in clinical trials to identify drug response patterns, novel combinations, and potential biomarkers of response for patient stratification in AML. Citation Format: Reecha Shah, Heba Wander, Michelle Hsueh, Muskan Floren, Wei Liu, Patricia Cheung, Courtney Andersen, Michael White, Nathan Kingston, Pablo Morentin Gutierrez, Lisa Drew, Giulia Fabbri, Elena Bibikova, Daniel Auclair. Multiplexed flow cytometry based immunophenotyping paired with functional ex vivo (MFLEX) drug profiling informs potential efficacy of therapeutic agents in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3904.

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3229.

Dismal outcome of refractory or relapsing patients with myelodysplasia-related acute myeloid leukemia partially alleviated by intensive chemotherapy.

Acute myeloid leukemia (AML) with myelodysplasia-related characteristics is a heterogeneous subset of AML that has been challenged throughout the history of myeloid malignancies classifications, considered to have similar outcomes as intermediate- or adverse-risk AML depending on the subgroup. However, little is known about the fate of these patients in refractory or relapsed situation (R/R) after first line therapy. A large series of R/R AML patients, recorded in the French DATAML registry, have received either intensive chemotherapy (ICT), azacitidine (AZA) as single agent, or best supportive care (BSC). A cohort of 183 patients (median age 63-year-old) with what was called at the time AML-MRC has been explored, and data are reported here. Patient status was refractory for 93, while 90 had relapsed. Respectively, 88, 34, and 61 were included in the three treatment arms. The median OS of the whole cohort was 4.2 months (95%CI: 3.1-5.6) with a mean 1-year overall survival of 24% ± 3.2%. There was no significant survival difference between refractory and relapsed patients. The BSC group had overall a significantly worse outcome (p = 0.0001), and this remained true in both refractory (p = 0.01) and relapsed (p = 0.002) patients. Similar survivals were observed in both groups comparing ICT and AZA. These data, reporting about an ill-explored population, indicate the poor prognosis of this condition where both ICT and AZA can be proposed. The latter, which was demonstrated here to be a feasible option, should be added to new targeted therapies.

Measuring response to therapy in AML: Difference from normal flow cytometry vs RQ-PCR.

Multiple technologies have been used to monitor response to therapy in acute myeloid leukemia (AML) to improve detection of leukemia over the standard of practice, morphologic counting of blasts. The two techniques most frequently used in a routine clinical setting, flow cytometry and RQ-PCR, differ in their targets, sensitivity, and ability to detect residual disease. Both flow cytometry and RQ-PCR detect the expression of abnormal gene products, at the protein level or RNA level, respectively. Flow cytometry can be applied to a broad range of AML cases while RQ-PCR is limited to specific genetic abnormalities identified in subsets of AML. This article compares the results when both techniques were used in a reference laboratory to monitor AML over the course of treatment, comparing quantitative and qualitative results.

A gain-of-function p53 mutant synergizes with oncogenic NRAS to promote acute myeloid leukemia in mice.

We previously demonstrated that a subset of acute myeloid leukemia (AML) patients with concurrent RAS pathway and TP53 mutations have an extremely poor prognosis and that most of these TP53 mutations are missense mutations. Here, we report that, in contrast to the mixed AML and T cell malignancy that developed in NrasG12D/+ p53-/- (NP-/-) mice, NrasG12D/+ p53R172H/+ (NPmut) mice rapidly developed inflammation-associated AML. Under the inflammatory conditions, NPmut hematopoietic stem and progenitor cells (HSPCs) displayed imbalanced myelopoiesis and lymphopoiesis and mostly normal cell proliferation despite MEK/ERK hyperactivation. RNA-Seq analysis revealed that oncogenic NRAS signaling and mutant p53 synergized to establish an NPmut-AML transcriptome distinct from that of NP-/- cells. The NPmut-AML transcriptome showed GATA2 downregulation and elevated the expression of inflammatory genes, including those linked to NF-κB signaling. NF-κB was also upregulated in human NRAS TP53 AML. Exogenous expression of GATA2 in human NPmut KY821 AML cells downregulated inflammatory gene expression. Mouse and human NPmut AML cells were sensitive to MEK and NF-κB inhibition in vitro. The proteasome inhibitor bortezomib stabilized the NF-κB-inhibitory protein IκBα, reduced inflammatory gene expression, and potentiated the survival benefit of a MEK inhibitor in NPmut mice. Our study demonstrates that a p53 structural mutant synergized with oncogenic NRAS to promote AML through mechanisms distinct from p53 loss.

Reduced Proteolipid Protein 2 promotes endoplasmic reticulum stress-related apoptosis and increases drug sensitivity in acute myeloid leukemia.

The Proteolipid Protein 2 (PLP2), a protein in the Endoplasmic Reticulum (ER) membrane, has been reported to be highly expressed in various tumors. Previous studies have demonstrated that the reduced PLP2 can induce apoptosis and autophagy through ER stress-related pathways, leading to a decreased proliferation and aggressiveness. However, there is no research literature on the role of PLP2 in Acute Myeloid Leukemia (AML). PLP2 expression, clinical data, genetic mutations, and karyotype changes from GEO, TCGA, and timer2.0 databases were analyzed through the R packages. The possible functions and pathways of cells were explored through GO, KEGG, and GSEA enrichment analysis using the clusterProfiler R package. Immuno-infiltration analysis was conducted using the Cibersort algorithm and the Xcell R package. RT-PCR and western blot techniques were employed to identify the PLP2 expression, examine the knockdown effects in THP-1 cells, and assess the expression of genes associated with endoplasmic reticulum stress and apoptosis. Flow cytometry was utilized to determine the apoptosis and survival rates of different groups. PLP2 expression was observed in different subsets of AML and other cancers. Enrichment analyses revealed that PLP2 was involved in various tumor-related biological processes, primarily apoptosis and lysosomal functions. Additionally, PLP2 expression showed a strong association with immune cell infiltration, particularly monocytes. In vitro, the knockdown of PLP2 enhanced endoplasmic reticulum stress-related apoptosis and increased drug sensitivity in THP-1 cells. PLP2 could be a novel therapeutic target in AML, in addition, PLP2 is a potential endoplasmic reticulum stress regulatory gene in AML.

Genetic and Pharmacological Interruption of the SIX1/EYA2 Axis Impairs Leukemia Proliferation

Background: The CALM-AF10 translocation is found in 5-10% of T-cell acute lymphoblastic leukemias (T-ALL), and a subset of acute myeloid leukemias (AML). CALM-AF10 leukemias are characterized by elevated expression of proleukemic HOXA genes. Since HOXA genes are difficult to target, we sought to identify non- HOXA effector genes through RNA sequencing and Microarray analyses, and showed that SIX1 expression is increased in CALM-AF10 leukemia cells. Like HOXA genes, SIX1 is a homeobox gene involved in cell proliferation, embryogenesis, and apoptosis. SIX1 interacts with the cofactor Eyes Absent 2 (EYA2), a protein phosphatase, to transcriptionally activate developmental genes, the TGFβ pathway, and the epithelial-mesenchymal transition (EMT). SIX1 expression is elevated during development but decreases with maturation. Intriguingly, overexpression of SIX1 has been observed in numerous malignancies including breast cancer, ovarian cancer, gliomas and esophageal cancer, and SIX1 is involved in accelerating the epithelial mesenchymal transition (EMT) and metastasis. Knockdown of either SIX1 or EYA2 has been shown to reverse these effects, and two novel inhibitors - one of the SIX1/EYA2 complex (Compound 8430, “8430”) and one of the EYA2 phosphatase (Compound 9987, “9987”) - have replicated the effects of SIX1 or EYA2 knockdown. Methods: SIX1 gene and protein expression were assessed in leukemia cell lines via RT-qPCR and Western Blot. SIX1 expression vectors were transduced into fetal liver hematopoietic stem cells (HSC) and immortalization was assessed using colony assays. CALM-AF10 leukemia cells lines were derived from irradiated mice transplanted with HSC retrovirally transfected with CALM-AF10 plasmids created in our lab. The established leukemia cell lines Jurkat, MOLT16, OCI-M2, TF-1, and SHI-1 were obtained from ATCC or DSMZ. shRNAs targeting SIX1 were lentivirally transduced into CALM-AF10 and SHI1leukemia cell lines. Cell-Titer-Glo assays and liquid culture were used to assess the effects of shRNAs, 8430, or 9987 on cell proliferation and cell growth. SIX1 gene expression in leukemias was determined from The Cancer Dependency Map from The Broad Institute using R. Results: We first validated RNAseq and Microarray studies, showing that CALM-AF10 induces SIX1 expression in murine fibroblasts and murine HSC, and localizes to the SIX1 locus. Furthermore, SIX1 overexpression was sufficient to immortalize murine fetal liver HSC (FL-mHSCs), implying a role in leukemic transformation. Multiple murine derived CALM-AF10 cell lines displayed increased SIX1 expression compared to fresh murine bone marrow and isolated HSCs. shRNA knockdown of SIX1 decreased viability of murine CALM-AF10 cells in culture. In addition, both the SIX1/EYA2 complex inhibitor (8430) and the EYA2 inhibitor (9987) decreased CALM-AF10 leukemia cell proliferation in vitro. To expand the relevance of SIX1 to other leukemias, we then queried The Cancer Dependency Map from the Broad Institute and identified other leukemia cell lines with increased SIX1 expression, including Jurkat and Molt16 (T-ALL), and SHI-1, OCIM2, and TF-1 (AML). SIX1 expression in these cells was validated by immunoblot and shRNA knockdown of SIX1 slowed the proliferation of SHI-1 cells. Finally, the 8430 and 9987 inhibitors reduced the proliferation of Jurkat, OCI-M2, and SHI-1 cells at doses similar to those used in CALM-AF10 leukemias. Conclusions: We have identified involvement of the SIX1/EYA2 axis in several different leukemias, including T-ALL and AML. Furthermore, we have shown that inhibitors of the SIX1/EYA2 interaction and of EYA2 reduce the proliferation of SIX1 expressing leukemias, suggesting a potential new therapeutic target in these leukemias.

Venetoclax Combined with Azacitidine and Homoharringtonine in Adults with Secondary Acute Myeloid Leukemia

Secondary acute myeloid leukemia is an aggressive subset of acute myeloid leukemia (AML) that is, in general, is associated with a very poor prognosis and high mortality. Venetoclax combined with hypomethylating agents have showed relatively successful therapeutic effective in patients with relapse/refractory AML, while efficacy is limited in high-risk subset AML. We hypothesised that Venetoclax combined with Azacitidine and Homoharringtonine could have improved activity in patients with secondary acute myeloid leukemia, particularly in high-risk subgroups. Methods This single center, phase 2 trial was done at the first affiliated hospital of Nanjing medical university. The study enrolled adult patients with diagnosed of secondary AML (AML progressed after preexisting myeloid neoplasms or treatment AML arising from prior exposure of leukemogenic therapies). Patients were required to have an Easter Cooperative Oncology Group performance status of 3 or less, and adequate end-organ function. Patients who had received previous BLC2 inhibitor therapy were excluded. Patients received Azacytidine 75mg/m 2 and Homoharringtonine 3mg for 7 days with venetoclax 400mg daily. The primary endpoint was overall response rate. The secondary endpoints include safety, overall survival, and duration of response (according to recommendations of European LeukemiaNet 2017 guidelines). The trial was registered on ClinicalTrial.gov (NCT 05513131) and continues to accrue patients. Results Since now, we enrolled 11patients; 8(72.7%) patients had secondary AML, 3 (27.3%) patients had treatment related AML, while 2(18.2%) patients had TP53 mutation. The median age was 62 years (48-78 years). At the July 30, 2023 data cutoff, median follow-up time was 36.7 weeks (range 6.57-82.29 weeks). The overall response rate was 81.8% (9 of 11 patients). The most common treatment-emergent adverse events included bone marrow suppression with grades 3 or 4 neutropenia (n=11/11) and febrile neutropenia (n=10/11). The 30-day mortality for all patients was 9.1% (n=1). 2(18.2%) patients were eligible for allogeneic hematopoietic stem cell transplantation. The median overall survival was not achieved. Conclusion Venetoclax combined with Azacitidine and Homoharringtonine regimen had a tolerable safety profile and showed encouraging clinical activity characterised by a high response rate. further patients are needed to validate these findings.

SIRT5 Regulates Energy Metabolism and Survival in Acute Lymphoblastic Leukemia Cells

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children, with a second incidence peak in adults over 60 years old. Long-term survival with currently available therapy regimens is >90% in children, but <50% in adults, reflecting differences in disease biology and tolerance of aggressive multi-agent chemotherapy regimens. While CAR-T cell therapy provides effective salvage to patients with relapsed B-cell ALL (B-ALL), outcome in relapsed T-ALL is exceptionally poor. In addition, therapy-related chronic functional impairment in pediatric ALL survivors are common. Less toxic, and more effective therapies are needed to improve ALL survival, particularly in adults, and reduce toxicity and functional impairment in all patients. There is evidence that energy restriction creates a barrier to malignant transformation as B cell precursors undergo somatic hypermutation and during T cell receptor diversification. Mutational inactivation of B-lineage transcription factors such as IKZF1 and PAX5 in B-ALL and aberrant expression of T-lineage regulators such as TLX1/3 in T-ALL disables this metabolic brake, promoting malignant transformation of B and T lymphoid precursors, respectively. Metabolism is being recognized as an attractive target in cancer therapy, but metabolic plasticity often limits the efficacy of targeting single metabolic pathways. SIRT5 is a lysine deacylase that regulates multiple metabolic pathways, including energy metabolism. We have previously reported that a subset of acute myeloid leukemia (AML) cases depends on SIRT5 as a master regulator of central energy metabolism, implicating SIRT5 as a therapy target in AML. We hypothesized that this metabolic vulnerability may extend to ALL. To study the function of SIRT5 in ALL, we stably expressed doxycycline (Dox)-inducible shRNAs targeting SIRT5 in ALL cell lines and primary samples. Knockdown (KD) of SIRT5 significantly inhibited growth and increased apoptosis in B-ALL and T-ALL cell lines. Similar results were seen with primary cells relapsed ALL, whereas SIRT5 KD had no effect on colony formation by normal CD34 + cord blood cells. We also injected mice with Jurkat T-ALL cells expressing Dox-inducible sh SIRT5 and luciferase. Dox-induced SIRT5 KD abrogated disease initiation and eliminated established disease (Figure 1A). To determine whether ALL cells require SIRT5 to maintain a metabolic state that is permissive to malignant transformation, we next evaluated the effect of SIRT5 disruption on energy metabolism, using Seahorse extracellular flux analysis. We found that SIRT5 deficient T-ALL cells had lower rates of both oxidative phosphorylation and glycolysis in comparison to SIRT5 wild type (WT) cells (Figure 1B). Next, to evaluate dependency of SIRT5-KD on multiple genotypes and therapy-resistant ALL, we engineered 30 ALL cell lines to stably express doxycycline sh SIRT5 representing B and T cell origin, diverse genotypes, and including drug resistant lines. We found that several B-ALL and T-ALL cell lines are highly dependent on SIRT5 for growth and survival, while others are not, showing a large range of sensitivity to SIRT5 KD. Therefore, to elucidate the potential mechanisms underlying SIRT5 dependence in ALL, we first analyzed whether SIRT5 KD differentially affects OXPHOS and glycolysis. Preliminary analysis of mitochondrial respiration upon SIRT5 KD suggested that OXPHOS and glycolysis are reduced in SIRT5-dependent cells, but not in SIRT5-independent cells upon SIRT5 KD. Finally, to determine whether Sirt5 disruption affects energy metabolism in normal hematopoietic stem cells (HSCs), we generated a tamoxifen-inducible conditional hematopoietic cell-specific Sirt5 knockout (KO) model. In preliminary experiments, we found no difference in mitochondrial respiration between normal HSCs with or without genetic absence of Sirt5, suggesting that leukemic cells, but not normal hematopoietic stem cells are dependent on Sirt5 for regulation of energy metabolism. Collectively, our results suggest that SIRT5 plays a critical oncogenic role in the development and maintenance of a subset of ALL, providing a rationale for the development of a clinical SIRT5 inhibitor. Targeting master regulators rather than single pathways may circumvent plasticity-based resistance in ALL.

Pre-Existing Myeloid Subclones May Account for a Subset of Acute Myeloid Leukemias and Myelodysplastic Neoplasms in Patients with Pre-Existing or Contemporary Lymphoid Disorders

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) are occasionally observed in association with lymphoid tumors. Most cases of myeloid disorders detected in patients with lymphoid tumors are attributed to chemotherapy, however, there is a subset of patients who receive a contemporary diagnosis of myeloid and lymphoid disease, and it is well known that, even in patients who were exposed to chemotherapy, the myeloid disease may both arise from clonal hematopoiesis substrate or harbor chemotherapy signature (Diamond, Blood 2023). Biological differences justified by the ontogenesis of the myeloid disease may justify prognosis and suggest appropriate intervention methods. Methods: We searched our patient database for AML and MDS which were diagnosed together with lymphoid diseases. Data was collected under the study NCT04298892, after approval of the ethical committee. Statistics were performed with Kaplan-Meyer estimation. Nonparametric tests were used as appropriate. Results: Out of 164 patients who were referred to our service from May 2021 to Jul 2023 for MDS or AML, 11 patients were diagnosed with AML or MDS together with a concomitant or previous lymphoid disease. In this population, the median age at AML or MDS diagnosis was 67 (range 58 - 82); 9/11 (82%) patients were male. Four out of 11 (36%) patients were diagnosed with AML, 7/11 with MDS (6 with blast < 5% and 1 with blast ≥ 5%). The diagnosis of myeloid and lymphoid diseases was concomitant in 2 patients (18%) and sequential in 9 patients (82%). The lymphoid diseases were smoldering multiple myeloma (3/11, 27%), multiple myeloma (5/11, 45%), diffuse large B-cell lymphoma (1/11, 9%), mantle cell lymphoma (1/11, 9%), marginal zone lymphoma (1/11, 9%). Eight out of 11 patients (73%) with lymphoid and myeloid disease had a history of chemotherapy before the diagnosis of MDS or AML; 5 patients (45%) were exposed to high-dose alkylating agents, 2 (18%) low-dose alkylating agents, 2 (18%) anthracyclines. Interestingly, 7 patients (64%) were diagnosed while they were receiving lenalidomide after a median exposure to lenalidomide of 8.8 months (4.9 - 30.73). In the 9 patients with consecutive lymphoid and myeloid disease diagnosis, AML or MDS were diagnosed after a median of 23 months (range 8 - 76; history of patients is plotted in figure 1). Regarding the AML or MDS, 3/11 patients (27%) had complex karyotype of which 1 had t(3;21)(q26;q22) RUNX1/MECOM rearrangement, 2/11 had TP53 mutation (both also had complex karyotype). The remaining patients mainly harbored methylation or splicing mutations and normal karyotype or karyotype single non-high-risk alterations. When compared, patients with complex alterations had similar times of onset from their first tumor and baseline characteristics; those patients however had an increased risk of death. Conclusions: The incidence of AML or MDS associated with lymphoid disease is low.Furthermore, the population is remarkably heterogeneous. The characteristics and the timing of onset of the disease account for different ontogenesis; a portion of the patient has a disease that harbors complex alterations, while the most frequent origin of AML and MDS can be researched in pre-existing or contemporary hematopoietic clones, that overall account for a whole bone-marrow disease. The relation between chemotherapy exposition and the biology of the AML or MDS is not univocal. This difference could be therapeutically relevant.