Primary Acute Myeloid Leukemia Research Articles

Leukemia and Lymphoma: An update of acute myeloid leukemia after Whole genome sequencing (WG)

Leukemia, known for several centuries, is a cancer of the blood and bone marrow. It starts when the DNA of a single cell in the bone marrow mutates and develops abnormal growth, which spill into blood stream. Based on the types, leukemia is classified in several groups for diagnosis and treatment. With the discovery of whole genome sequences (WGS), scientists are attempting to find out accurate diagnosis and treatment. Primary Acute myeloid leukemia (AML) samples are feasible and can detect novel, clinically relevant mutations including the clonal heterogeneity of this disease and clonal evolution that occurs over time. Some of the novel mutations are highly recurrent (>20% of patients), but there appears to be a continuum of mutation frequency down to rare (<5%) or even singleton mutations that may be relevant for the biology of this disease. Large cohorts of well-annotated samples are needed to establish mutation frequencies, implicate biological pathways, and demonstrate genotype: phenotype correlations. Recent advances in genomic techniques have unraveled the molecular complexity of AML leukemogenesis, which in turn have led to refinement of risk stratification and personalized therapeutic strategies for patients with AML

AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

A chemically adjustable BMP6-IL6 axis in mesenchymal stem cells drives acute myeloid leukemia cell differentiation

Chemotherapy alone or in combination with allogeneic stem cell transplantation has been the standard of care for acute myeloid leukemia (AML) for decades. Leukemia relapse with limited treatment options remains the main cause of treatment failure. Therefore, an effective and safe approach to improve treatment outcomes is urgently needed for most AML patients. Mesenchymal stem cells (MSCs) have been reported to efficiently induce apoptosis and shape the fate of acute myeloid leukemia cells. Here, we identified LG190155 as a potent compound that enhances the antileukemia efficiency of MSCs. Pretreatment of MSCs with LG190155 significantly provoked differentiation in both AML patient-derived primary leukemia cells and AML cell lines and reduced the tumor burden in the AML mouse model. Using the quantitative proteomic technique, we discovered a pivotal mechanism that mediates AML cell differentiation, in which autocrine bone morphogenetic protein 6 (BMP6) in MSCs boosted IL-6 secretion and further acted on leukemic cells to trigger differentiation. Furthermore, the activity of the BMP6-IL6 axis was dramatically enhanced by activating vitamin D receptor (VDR) in MSCs. Our data illustrated an effective preactivated approach to reinforcing the antileukemia effect of MSCs, which could serve as an effective therapeutic strategy for AML.

Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML.

Loss of the stress sensor GADD45A promotes stem cell activity and ferroptosis resistance in LGR4/HOXA9-dependent AML

AbstractThe overall prognosis of acute myeloid leukemia (AML) remains dismal, largely because of the inability of current therapies to kill leukemia stem cells (LSCs) with intrinsic resistance. Loss of the stress sensor GADD45A is implicated in poor clinical outcomes, but its role in LSCs and AML pathogenesis is unknown. Here, we define GADD45A as a key downstream target of LGR4 oncogenic signaling and discover a regulatory role for GADD45A loss in promoting leukemia-initiating activity and oxidative resistance in LGR4/HOXA9-dependent AML, a poor prognosis subset of leukemia. Knockout of GADD45A enhances AML progression in murine and patient-derived xenograft (PDX) mouse models. Deletion of GADD45A induces substantial mutations, increases LSC self-renewal and stemness in vivo, and reduces levels of reactive oxygen species (ROS), accompanied by a decreased response to ROS-associated genotoxic agents (eg, ferroptosis inducer RSL3) and acquisition of an increasingly aggressive phenotype on serial transplantation in mice. Our single-cell cellular indexing of transcriptomes and epitopes by sequencing analysis on patient-derived LSCs in PDX mice and subsequent functional studies in murine LSCs and primary AML patient cells show that loss of GADD45A is associated with resistance to ferroptosis (an iron-dependent oxidative cell death caused by ROS accumulation) through aberrant activation of antioxidant pathways related to iron and ROS detoxification, such as FTH1 and PRDX1, upregulation of which correlates with unfavorable outcomes in patients with AML. These results reveal a therapy resistance mechanism contributing to poor prognosis and support a role for GADD45A loss as a critical step for leukemia-initiating activity and as a target to overcome resistance in aggressive leukemia.

Abstract LB026: Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics

Abstract In the ongoing pursuit of addressing the formidable challenge presented by Acute Myeloid Leukemia (AML) treatment, this comprehensive study delves into the intricate antileukemic effects of Ful-23, our newly developed Monocarboxylate Transporter 4 (MCT4) inhibitor. Positioned as a promising candidate, Ful-23's unique mechanisms and potential synergies with established chemotherapeutic agents, such as Cytarabine, are elucidated in this research. Intracellular alkalinization stands as a crucial factor contributing to the evasion of chemotherapy by cancer cells. It serves as the primary determinant for the heightened resistance exhibited by cancer cells against chemotherapy interventions. The investigation reveals the multifaceted impact of Ful-23 on AML cells, showcasing its capacity to induce intracellular lactate accumulation and acidification. These metabolic alterations correlate with a substantial inhibition of proliferation, as evidenced by an IC50 of approximately 0.55 μM, and a marked induction of apoptosis, particularly notable in the MV411 AML cell line. These findings underscore Ful-23's potential as a potent antileukemic agent with distinct cellular effects. A collaborative approach involves the combination of low concentrations of Ful-23 (0.312 μM) with Cytarabine (0.312 μM), revealing a significant enhancement in cytarabine-induced proliferation inhibition and apoptosis when Ful-23 is introduced concurrently. Importantly, the translational impact of these findings extends to a zebrafish model, utilizing both MV411 cells and transplanted primary AML patient-derived bone marrow cells. In the zebrafish AML model, the combination treatment of Ful-23 and Cytarabine demonstrates a significant enhancement in their respective antitumor effects compared to monotherapy, indicating a pronounced synergistic effect. This additional validation strengthens the clinical relevance of the observed synergistic antileukemic interactions between Ful-23 and Cytarabine. Essentially, this investigation advances our understanding of AML treatment dynamics, emphasizing the crucial role of MCT4 inhibition and elucidating the mechanism by which it leads to intracellular acidification in synergistic therapeutic approaches for this intricate and formidable disease. Citation Format: Yong Wu, Ke Wu, Jay Vadgama. Exploring MCT4 inhibitor Ful-23 and cytarabine synergy in acute myeloid leukemia therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB026.

Targeting CD33+ acute myeloid leukemia with GLK-33, a lintuzumab-auristatin conjugate with a wide therapeutic window.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of AML blasts, making it an attractive target for therapy of acute myeloid leukemia (AML). While previous CD33-targeting antibody-drug conjugates (ADCs) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novel ADC with improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linker-payloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated anti-tumor activity at single dose as low as 300 µg/kg in mice, while maintaining tolerability at single dose of 20 - 30 mg/kg in rats. In contrast to both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials.

Combining CRISPR-Cas9 and TCR exchange to generate a safe and efficient cord blood-derived T cell product for pediatric relapsed AML

BackgroundHematopoietic cell transplantation (HCT) is an effective treatment for pediatric patients with high-risk, refractory, or relapsed acute myeloid leukemia (AML). However, a large proportion of transplanted patients eventually die due...

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Identification of Nonfunctional Alternatively Spliced Isoforms of STING in Human Acute Myeloid Leukemia.

We find that AML acquires resistance to innate immune activation via the STING pathway through aberrant splicing of the STING transcript including two novel forms described herein that act as dominant negatives. These data broaden understanding of how cancers evolve STING resistance, and suggest that the AML tumor microenvironment, not the cancer cell, should be the target of therapeutic interventions to activate STING.

Abstract 44: A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells

Abstract Chimeric antigen receptor (CAR) T cell therapies for hematologic malignancies, while promising and efficacious, can also result in a variety of adverse effects. Of note, on-target, off-tumor toxicity is one adverse effect that continues to limit the safety and efficacy of CAR-T cells and has even led to life-threatening or fatal outcomes in patients (Morgan et al. 2010.). To address this, we have developed a unique, codon-based approach by replacing strategically selected codons within CAR non-antigen binding domains with their rare synonymous isoforms. Hoekema et al. (1987) reported the ability of synonymous rare codons to alter gene and protein expression in other model systems. In CAR-T cells, we aim to use this novel, codon-based approach to fine tune and modulate CAR-T cell expression and function to elicit full on-tumor activity while lessening off-tumor toxicity. In our approach, we replaced specific endogenous codons within the non-antigen binding domains of Hu8F4-CAR, a TCR-like 2nd generation CAR developed by our group which targets the leukemia-associated antigen PR1/HLA-A2 (Ma et al. 2016.). Briefly, we created variants of Hu8F4-CAR containing rare codon substitutions in two subtypes: intracellular-based (type 1) and combined extracellular- and intracellular-based (type 2). Each variant subtype (type 1 or 2) had either between 6 and 30 or 27 and 57 rare codons introduced, respectively. Expression profiling and functional assessment of the Hu8F4-CAR variants was assessed in the Jurkat mutant cell line (J.RT3-T3.5) or human primary healthy peripheral blood mononuclear cells (PBMCs) that were enriched for T cells, respectively. Expression profiling results show that surface expression is reduced in J.RT3-T3.5 cells when assessed by flow cytometry by 1-1.5-fold compared to unmodified Hu8F4-CAR. When evaluated by immunoblotting, novel lower molecular weight bands emerge only in rare codon-modified variants, suggesting that rare codons may have altered translational or post-translational processing leading to truncated CAR protein isoforms (30, 45, or 60kDa vs 73kDa for unmodified Hu8F4-CAR), which may contribute to reduced cell surface CAR expression. Furthermore, we have conducted in vitro functional testing of the rare codon-modified variants and show reduced activity against non-malignant off-tumor targets that express PR1 and/or HLA-A2 including healthy donor PBMCs, mobilized hematopoietic cells, and bone marrow. Importantly, in addition to reduced off-tumor activity against healthy myeloid cells, rare codon-modified variants preserve their activity against on-tumor target cells, including the U937-A2+ cell line and primary acute myeloid leukemia. Altogether, these results suggest that our approach can modulate CAR expression and functionality creating a novel methodology to reduce the likelihood of off-tumor adverse effects typically associated with CAR therapies. Citation Format: Rolando A. Vedia, Sijie Lu, Anne V. Philips, Anna Sergeeva, Lisa S. St John, Karen Clise-Dwyer, Gheath Alatrash, Jeffrey J. Molldrem. A novel, codon-based method to modulate off-tumor toxicity of chimeric antigen receptor (CAR) T cells [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 44.

Abstract 2548: Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples

Abstract Background: Assessing drug efficacy in primary acute myeloid leukemia (AML) patient cells is crucial for pinpointing drug targets and rationally selecting therapies for patients. We have previously shown that our flow cytometry-based predictive precision medicine platform (PPMP) predicts patient outcomes for standard-of-care (SOC) AML treatments using fresh blood or bone marrow samples with high precision. Here, we wanted to test the feasibility and efficacy of the PPMP on cryopreserved patient-derived leukemic samples. However, cryopreserved leukemic samples are known for exhibiting low viability and cell recovery, rendering their practical utilization often challenging. In this study, we adapt an existing methodology for cryopreservation for patient-derived leukemic cells to investigate the potential of cryopreserved samples as an alternative to fresh samples for ex vivo prediction of treatment outcomes on our PPMP. Methods: We screened freshly isolated white blood cells from AML patients on the PPMP to assess their response to multiple SOC single agents and combinations. The assay was also run without drug treatments immediately after receiving the fresh sample (≤ 2 days from draw) to establish a phenotype baseline. The cryopreserved samples were processed using a similar protocol, with post-thaw culture times of either 24 or 48 hours in two types of cytokine-containing media. A comparative analysis between untreated fresh and cryopreserved samples determined the optimal post-thaw culture time for cryopreserved samples. Once culture time was optimized, we screened cryopreserved samples on the PPMP with identical SOC drug conditions as the fresh samples and compared the response of leukemic blasts to drug treatment as well as impacts on other phenotypic markers. Results: Based on our preliminary data, we found that culturing cryopreserved cells for 24 hours post-thaw was sufficient to maintain a minimum cell viability of 80% while closely maintaining a phenotype that recapitulated that of the corresponding fresh samples. In response to drug treatments within the ex vivo assays, preliminary data support a correlation (Decitabine single agent, R2=0.81) between the response profiles of cryopreserved and fresh patient samples as gauged from AUC calculations. Conclusion: While the preferred method for screening is to use fresh patient samples, many practical constraints make it challenging or unfeasible. The ability of Notable Lab’s high-throughput platform to use cryopreserved samples opens the doors to enhanced retrospective studies leveraging clinically annotated data from biobanks or clinical trials. These retrospective studies provide a substantial reduction in the time needed to conclude such investigations. Citation Format: Lokesh Patil, Christine J. Gu, Wade Anderson, Nicola Long, Kara Johnson, Cristina Tognon, Markus D. Lacher. Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples [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 2548.

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against 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 618.

Abstract 6341: Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE

Abstract Background: Myeloid epithelial reproductive tyrosine kinase (MERTK), a transmembrane protein receptor from the TAM (Tyro3, Axl, Mertk) family, is overexpressed in cancer and associated with reduced apoptosis and chemoresistance, making it an attractive therapeutic target. RGX-019-MMAE, a novel humanized IgG1-MMAE antibody-drug conjugate (ADC) (Inspirna, Inc), selectively binds to MERTK with high affinity on tumor cells, resulting in internalization and degradation of the receptor. It then induces cytotoxicity through the release of the payload, MMAE (monomethyl auristatin E), which disrupts mitosis. We hypothesize that targeting MERTK via RGX-019-MMAE exerts an antileukemic effect. Methods: First, to determine the clinical relevance of MERTK, using the Oregon Health and Science University (OHSU) acute myeloid leukemia (AML) dataset, we divided patients into low and high MERTK mRNA expression based on mean expression and compared their overall survival. To identify the AML subtypes with the highest MERTK expression, we analyzed samples from 810 AML patients at MD Anderson using reverse phase protein arrays (RPPA), as well as 8 AML cell lines and the peripheral blood of 9 AML patients using flow cytometry. Then, the cell lines with the highest MERTK expression were treated with varying doses of RGX-019-MMAE or isotype control for 120 hours and assessed for viability with CellTiter-Glo 2.0. Likewise, we administered RGX-019-MMAE to primary cells from AML patients and determined the anti-leukemic effect. Finally, we investigated the synergistic effect of RGX-019-MMAE with venetoclax, a BCL2 inhibitor, or cytarabine, a chemotherapy agent, in vitro. Results: In the AML OHSU dataset, patients with high MERTK mRNA expression had significantly worse overall survival (p=0.02). RPPA revealed that patients with PTPN11 mutation, t (9;11), and monocytic AML subtypes had significantly higher MERTK protein expression. MERTK protein expression varied in AML cell lines and was highest in Kasumi-1 and OCI-AML3. Treatment of these two cell lines with RGX-019-MMAE resulted in significantly more killing of leukemic cells than the isotype control ADC in a dose-dependent manner (p&amp;lt;0.01). Similarly, in primary AML cells with high MERTK expression, RGX-019-MMAE treatment induced significant cell death compared to the control ADC. In addition, Kasumi-1 and OCl-AML3 cells treated with RGX-019-MMAE combined with venetoclax or cytarabine showed a synergistic cytotoxic effect in a dose-dependent manner. Conclusion: High MERTK expression in monocytic AML suggested that MERTK is a promising therapeutic target in this subtype. RGX-019-MMAE significantly induced AML cell death and produced a synergistic anti-leukemic effect in combination with chemotherapeutic agents and targeted therapy in vitro, suggesting its potential for targeting patients with high-risk AML. Citation Format: Anudishi Tyagi, Maryam Siddiqui, Isabel Kurth, Shugaku Takeda, Amanda Eckstrom, Jenny Borgman, Abhishek Maiti, Venkata Lokesh Battula. Targeting myeloid epithelial tyrosine kinase (MERTK) receptor in acute myeloid leukemia using a novel antibody-drug conjugate, RGX-019-MMAE [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 6341.

Abstract 2104: Targeting stem cell proteostasis pathways sensitizes acute myeloid leukemia to proteasome inhibitors

Abstract Proteasome inhibitors are highly effective for treating multiple myeloma, but exhibit little efficacy in other blood cancers. In this study, we set out to determine why acute myeloid leukemia (AML) is largely refractory to proteasome inhibition and if we could sensitize human AML to this class of drugs. Efficacy of proteasome inhibitors in myeloma partly depends on disruption of protein homeostasis (proteostasis). This is driven by an accumulation of misfolded proteins that activate the unfolded protein response (UPR) and integrated stress response (ISR). Due to their highly secretory nature, myeloma cells exhibit relatively elevated baseline endoplasmic reticulum stress, and are thus sensitive to proteasome inhibition as they readily activate a terminal UPR. However, we found that proteasome inhibition fails to significantly disrupt proteostasis in human AML cells. We recently discovered that hematopoietic stem cells utilize distinct pathways to regulate proteostasis, and we hypothesized that AML cells co-opt stem cell proteostasis pathways to cope with proteasome inhibition and preserve proteostasis. To test this, we first performed RNA-sequencing which revealed that AML cells respond to proteasome inhibition by activating two distinct proteostasis pathways normally utilized by hematopoietic stem cells: autophagy and the heat shock response. Concurrent proteasome and autophagy inhibition significantly increased unfolded protein content in multiple human AML cell lines. This proteostasis disruption was sufficient to induce a terminal ISR marked by activation of the eIF2α-ATF4-CHOP signaling axis that severely attenuated protein synthesis, reduced proliferation, and induced widespread apoptosis in human AML cell lines and primary AML patient samples. In contrast to AML cells, dual proteasome and autophagy inhibition was well tolerated by healthy human CD34+ hematopoietic stem and progenitor cells, suggesting a tractable therapeutic window for treating AML. Indeed, dual proteasome and autophagy inhibition was tolerated in vivo, and decreased leukemia burden in mice xenografted with human AML cell lines. Similar to autophagy inhibition, disabling the heat shock response by deleting Heat shock factor 1 (HSF1) sensitized human AML cell lines to proteasome inhibitors. Treatment of HSF1-deficient AML cells with a proteasome inhibitior induced a significant increase in unfolded protein, activated the ISR, reduced growth and protein synthesis, and led to severe apoptosis. Concurrent HSF1 deletion and proteasome inhibition dramatically reduced AML disease burden in vivo and extended median survival from 40 to 140 days. Overall, this study revealed that AML cells hijack stem cell proteostasis pathways to promote growth, survival, and therapeutic resistance. Targeting the unique configuration of the proteostasis network is uncovering new therapeutic strategies to eliminate AML. Citation Format: Kentson Lam, Yoon Joon Kim, Carlo M. Ong, Fanny J. Zhou, Andrea Z. Liu, Bernadette A. Chua, Jie-Hua Zhou, Edward D. Ball, Robert A. Signer. Targeting stem cell proteostasis pathways sensitizes acute myeloid leukemia to proteasome inhibitors [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 2104.

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 2081: Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials

Abstract Dysregulated RNA splicing is a molecular feature that characterizes almost all tumor types. Splicing is altered in cancer owing to both recurrent mutations and deregulated expression of trans-acting factors that catalyze and regulate splicing. Therefore, splicing can be targeted for the development of new drugs to treat cancers, particularly those with mutations in splicing factors. In myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), genes that encode splicing factors are among those with the highest mutation rates. These mutations are heterozygous and largely mutually exclusive, suggesting the possibility of synthetic lethality due to multiple splicing factor mutations. Such synthetic lethality can be applied as an innovative strategy to develop drugs for the treatment of MDS and AML. We have reported the development of a CLK kinase inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. The mechanism of action of CTX-712 involves the suppression of the phosphorylation of multiple SR proteins, including SRSF2/3/4/6, alteration of nuclear speckle morphology, and switching of binding partners of SRSF2. RNA-seq analysis revealed that CTX-712 induced global alterations in splicing, typically resulting in exon exclusion (exon skipping) of cassette exons. Notably, the anti-leukemic effects of CTX-712 positively correlated with the degree of altered splicing, suggesting that CTX-712 inhibits RNA splicing. [Cancer Res (2022) 82 (12_Supplement): 5494., Blood (2022) 140 (Supplement 1): 489-490]. However, patient stratification markers for CTX-712 based on the above mechanism are still undefined. To identify biomarkers for CTX-712, we performed a comprehensive analysis by integrating both wet and dry DNA/RNA sequencing on more than 60 ex vivo primary samples from MDS/AML patients and 23 MDS/AML-derived xenografts (PDX). Unexpectedly, regardless of their splicing factor-mutation status, various types of primary AML cells and PDX models were highly sensitive to CTX-712. Both the primary cells and PDX models showed significant responses to CTX-712 and greater changes in splicing. Additionally, we performed biomarker analysis by comparing gene mutations and RNA splicing patterns between the sensitive and insensitive models and associating them with the anti-tumor effect. We identified several potential biomarkers that can indicate sensitivity to CTX-712. Mechanistic studies that aim to determine why these biomarkers could make cancer more sensitive to splicing perturbation by CTX-712 are ongoing. In conclusion, our results demonstrated a significant effect of CTX-712 on MDS/AML-derived models regardless of their splicing factor-mutation status. Additionally, we present novel biomarkers that could predict sensitivity to CTX-712. Citation Format: Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, Hirokazu Tozaki, Yoshihiko Satoh, Takuto Mori, June Takeda, Hideki Makishima, Masahiro Nakagawa, Yasuhito Nannya, Seishi Ogawa. Biomarkers for CLK inhibitor CTX-712 treatment response in myeloid neoplasms: Paving the way toward clinical trials [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 2081.

Abstract 6322: CCTx-001 CAR T-cells targeting the AML specific antigen IL-1RAP show robust pre-clinical anti-tumor efficacy

Abstract Despite significant advancements in acute myeloid leukemia (AML) treatment, patients with relapsed/refractory (r/r) AML have limited therapeutic options. Novel approaches are urgently needed. CAR T-cell therapy has shown remarkable success in hematological malignancies. However, its application in AML has been hampered by the absence of highly AML-specific antigens, making current CAR T-cell AML therapies under development poorly tolerated and effective. This pre-clinical study introduces a novel CAR T-cell therapy, CCTx-001, specifically designed to target IL-1RAP, a 78-kDa transmembrane glycoprotein. IL-1RAP, is highly expressed on AML cells but not on healthy hematopoietic stem and progenitor cells (CD34+ HSPC). Additionally, transcriptomic analyses reveal a correlation between high IL-1RAP expression and poor prognosis in AML patients. IL-1RAP's predominant presence on myeloid leukemia cells, coupled with their dependence to IL-1RAP-related pathways for their survival, presents a promising avenue for CAR T-cell therapy without the risk of treatment-induced antigen loss. CCTx-001, an autologous CAR T-cell therapy, was developed to target IL-1RAP on AML cells. It comprises CD8+ and CD4+ T-cells transduced with a highly specific anti-IL-1RAP Single-Chain Variable Fragment (scFv), an IgG1 hinge region, CD28, 4-1BB co-stimulatory domains, and a CD3ζ activation domain. In vitro experiments confirmed CCTx-001's efficacy in killing AML cell lines and primary AML cells of varying IL-1RAP expression levels, also demonstrating cytotoxicity and cytokine release that was dependent on the (E:T) ratio and target expression. Furthermore, in vivo studies using immunocompromised NCG mice confirmed CCTx-001's ability to target IL-1RAP and inhibit the growth of IL-1RAP-positive human AML tumor xenografts, leading to increased survival time. Biodistribution analyses confirmed the successful adoptive transfer, expansion and persistence of CCTx-001 CAR T-cells in vivo, only when injected into IL-1RAP-expressing tumor-bearing mice, which correlated with reduced levels of circulating AML cells. The promising preclinical data presented herein warrant the evaluation of CCTx-001 CAR T-cell therapy in clinical trials for r/r AML patients. CCTx-001 CAR T-cell therapy offers a potential breakthrough in addressing the unmet medical needs of AML patients with resistant or refractory disease. Citation Format: Edouard De Dreuzy, Soha Reda El Sayed, Delphine Genin, Clementine Primus, Lucie Bouquet, Alexis Collette, Olivier Favre-Bulle, Erik Rutjens, Jens Hasskarl, Ksenija Pavletic, Marina Deschamps, Christophe Ferrand, Richard C. A. Sainson. CCTx-001 CAR T-cells targeting the AML specific antigen IL-1RAP show robust pre-clinical anti-tumor efficacy [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 6322.

Abstract 5178: Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients

Abstract INTRODUCTION: Volasertib, a polo-like kinase 1 (PLK1) inhibitor, has a roughly 30% CR/CRi response rate in de novo acute myeloid leukemia (AML) patients when combined with low-dose cytarabine. However, its development was halted due in part to toxicity in a Phase 3 study. We have built a flow cytometry-based, high-throughput predictive precision medicine platform to forecast clinical responses to various treatments. Here, we present our design plan for a Phase 2a clinical trial with volasertib in relapsed/refractory (R/R) AML using a modified protocol to reduce toxicity and the development of a companion diagnostic to predict responders in a follow-up Phase 2b study. METHODS: We will initiate an open-label clinical trial in AML with volasertib in venetoclax-hypomethylating agent (HMA) R/R patients while co-developing a companion diagnostic to selectively enroll predicted responders in a follow-up, Phase 2b, study. Primary and secondary objectives will focus on efficacy/safety of intravenous volasertib and ex vivo responses, respectively. To reduce the toxicity seen in the Phase 3 study, specific strategies like tailored dosing will be implemented. To develop the clinical trial assay, peripheral blood or bone marrow samples were collected from AML patients and treated ex vivo with volasertib for 3 days. Surviving blast cells were enumerated by flow cytometry. Using a dynamic separation model restricted to the 35% most sensitive samples, we estimated optimal blast cutoffs for volasertib sensitivity. RESULTS: In single-agent experiments with 41 primary AML samples (31 de novo, 9 R/R, 1 with disease status unclear), we observed dose-response profiles consistent with volasertib’s role as a cell cycle inhibitor. Irrespective of the disease status, median and mean EC50 values were about 10 nM (1st-3rd quartile from 7-12 nM). To identify an optimal concentration of volasertib for patient stratification, we compared the ex vivo resistant blast fractions at each concentration with the areas under the dose-response curves (AUCs), assuming that in our case, AUCs most accurately reflect clinical responses. At 31.6 and 100 nM, the Pearson correlation coefficients were highest (r &amp;gt; 0.8), suggesting that these are optimal stratifying doses. With these concentrations and corresponding blast cutoff values, 32-33% of the de novo and 25% of the R/R patients would have been predicted to be responders were they given volasertib, assuming a positive predictive value of 100%. CONCLUSION: Volasertib’s EC50 values clustered within a narrow range, suggesting that EC50 may not be a suitable metric to stratify patients into responders and nonresponders, in contrast to AUCs and residual blast fractions at 31.6 and 100 nM of volasertib, which displayed more heterogeneous distributions. Limitations include small sample sizes, especially for the R/R group. Citation Format: Markus Daniel Lacher, Glenn Michelson, Allen Ling, Wade Anderson, Jeaneille Vinas, Christine J. Gu, Chris Leonardi, Joseph Wagner, Gabriel N. Mannis. Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients [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 5178.