Primary Acute Myeloid Leukemia Cells Research Articles

The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

Selective degradation of mutant FMS-like tyrosine kinase-3 requires BIM-dependent depletion of heat shock proteins

AbstractInternal tandem duplications in the FMS-like tyrosine kinase-3 (FLT3-ITD) are common mutations in acute myeloid leukemia (AML). Proteolysis-targeting chimeras (PROTACs) that induce proteasomal degradation of mutated FLT3 emerge as innovative pharmacological approach. Molecular mechanisms that control targeted proteolysis beyond the ubiquitin-proteasome-system are undefined and PROTACs are the only known type of FLT3 degraders. We report that the von-Hippel-Lindau ubiquitin-ligase based FLT3 PROTAC MA49 (melotinib-49) and the FLT3 hydrophobic tagging molecule MA50 (halotinib-50) reduce endoplasmic reticulum-associated, oncogenic FLT3-ITD but spare FLT3. Nanomolar doses of MA49 and MA50 induce apoptosis of human leukemic cell lines and primary AML blasts with FLT3-ITD (p < 0.05-0.0001), but not of primary hematopoietic stem cells and differentiated immune cells, FLT3 wild-type cells, retinal cells, and c-KIT-dependent cells. In vivo activity of MA49 against FLT3-ITD-positive leukemia cells is verified in a Danio rerio model. The degrader-induced loss of FLT3-ITD involves the pro-apoptotic BH3-only protein BIM and a previously unidentified degrader-induced depletion of protein-folding chaperones. The expression levels of HSP90 and HSP110 correlate with reduced AML patient survival (p < 0.1) and HSP90, HSP110, and BIM are linked to the expression of FLT3 in primary AML cells (p < 0.01). HSP90 suppresses degrader-induced FLT3-ITD elimination and thereby establishes a mechanistically defined feed-back circuit.

The tandem CD33-CLL1 CAR-T as an approach to treat acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by high heterogeneity, poor long-term survival, and a propensity for relapse. Exceptional efficacy in treating recurrent or refractory B-lymphoid malignancies has been demonstrated by Chimeric antigen receptor T cells (CAR-T cells). Given the therapeutic potential of targeting both CD33 and C-type lectin-like molecule-1 (CLL1) in AML, the development of a dual-targeting CD33-CLL1 CAR-T cells assumes significant importance. The expressions of CD33 and CLL-1 antigens in peripheral blood cells and bone marrow cells from AML patients was assessed. Subsequently, a Chimeric Antigen Receptor (CAR) incorporating a dual-specific single-chain variable fragment targeting CLL1 and CD33 (CD33-CLL1-CAR-T) was engineered. The anti-tumor efficacy and potential side effects of CD33-CLL1-CAR-T cells were comprehensively investigated in both in vitro and in vivo settings. The constructed tandem CD33-CLL1 CAR-T exhibited potent cytotoxicity against leukemia cell lines and human primary AML cells in vitro. Co-cultivation of AML blasts with CD33-CLL1-CAR-T cells resulted in effective proliferation and the secretion of substantial quantities of GM-CSF and IFN-γ. Importantly, the impact of CD33-CLL1-CAR-T cells on normal hematopoietic stem cells was minimal, ensuring safety in vivo mouse models. Notably, significant anti-leukemic activity was observed in the mouse model, with CD33-CLL1-CAR-T cells leading to tumor eradication and prolonged survival. The tandem CD33-CLL1 CAR-T cells not only efficiently eliminated AML blasts but also exhibited low cytotoxicity toward normal hematopoietic stem cells (HSCs). These findings underscore the potential clinical applicability of the tandem CD33-CLL1 CAR-T cells as an effective and safe treatment strategy for AML, representing a noteworthy advancement in the field of CAR-T cells therapy.

Genome-wide CRISPR screening identifies critical role of phosphatase and tensin homologous (PTEN) in sensitivity of acute myeloid leukemia to chemotherapy.

Although significant progress has been made in the development of novel targeted drugs for the treatment of acute myeloid leukemia (AML) in recent years, chemotherapy still remains the mainstay of treatment and the overall survival is poor in most patients. Here, we demonstrated the antileukemia activity of a novel small molecular compound NL101, which is formed through the modification on bendamustine with a suberanilohydroxamic acid (SAHA) radical. NL101 suppresses the proliferation of myeloid malignancy cells and primary AML cells. It induces DNA damage and caspase 3-mediated apoptosis. A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) library screen revealed that phosphatase and tensin homologous (PTEN) gene is critical for the regulation of cell survival upon NL101 treatment. The knockout or inhibition of PTEN significantly reduced NL101-induced apoptosis in AML and myelodysplastic syndrome (MDS) cells, accompanied by the activation of protein kinase B (AKT) signaling pathway. The inhibition of mammalian target of rapamycin (mTOR) by rapamycin enhanced the sensitivity of AML cells to NL101-induced cell death. These findings uncover PTEN protein expression as a major determinant of chemosensitivity to NL101 and provide a novel strategy to treat AML with the combination of NL101 and rapamycin.

Microenvironment matters: In vitro 3D bone marrow niches differentially modulate survival, phenotype and drug responses of acute myeloid leukemia (AML) cells

Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34−phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.

A dual-targeting approach with anti-IL10R CAR-T cells engineered to release anti-CD33 bispecific antibody in enhancing killing effect on acute myeloid leukemia cells.

Immunotherapies, including chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs), encounter several challenges in the management of acute myeloid leukemia (AML), including limited persistence of these treatments, antigen loss and resistance of leukemia stem cells (LSCs) to therapy. Here, we proposed a novel dual-targeting approach utilizing engineered anti-IL10R CAR-T cells to secrete bispecific antibodies targeting CD33. This innovative strategy, rooted in our previous research which established a connection between IL-10 and the stemness of AML cells, designed to improve targeting efficiency and eradicate both LSCs and AML blasts. We first demonstrated the superior efficacy of this synergistic approach in eliminating AML cell lines and primary cells expressing different levels of the target antigens, even in cases of low CD33 or IL10R expression. Furthermore, the IL10R CAR-T cells that secret anti-CD33 bsAbs (CAR.BsAb-T), exhibited an enhanced activation and induction of cytotoxicity not only in IL10R CAR-T cells but also in bystander T cells, thereby more effectively targeting CD33-positive tumor cells. Our in vivo experiments provided additional evidence that CAR.BsAb-T cells could efficiently redirect T cells, reduce tumor burden, and demonstrate no significant toxicity. Additionally, delivering bsAbs locally to the tumor sites through this strategy helps mitigate the pharmacokinetic challenges typically associated with the rapid clearance of prototypical bsAbs. Overall, the engineering of a single-vector targeting IL10R CAR, which subsequently secretes CD33-targeted bsAb, addresses the issue of immune escape due to the heterogeneous expression of IL10R and CD33, and represents a promising progress in AML therapy aimed at improving treatment outcomes.

Targeting HMGCS1 restores chemotherapy sensitivity in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common hematological malignancy with overall poor prognosis. Exploring novel targets is urgent and necessary to improve the clinical outcome of relapsed and refractory (RR) AML patients. Through clinical specimens, animal models and cell-level studies, we explored the specific mechanism of 3-hydroxy-3-methylglutaryl coenzyme A synthase 1 (HMGCS1) in AML and the mechanism of targeting HMGCS1 to attenuate cell proliferation, increase chemotherapy sensitivity and improve the occurrence and development of AML. Here, we reveal that HMGCS1 is overexpressed in RR patients and negatively related to overall survival (OS). Knocking out HMGCS1 in AML cells attenuated cell proliferation and increased chemotherapy sensitivity, while stable overexpression of HMGCS1 had the opposite effects. Mechanistically, we identified that knockout of HMGCS1 suppressed mitogen-activated protein kinase (MAPK) pathway activity, while overexpression of HMGCS1 could remarkably enhance the pathway. U0126, a MEK1 inhibitor, offset the effects of HMGCS1 overexpression, indicating that HMGCS1 promotes RR AML through the MAPK pathway. Further, we verified that hymeglusin, a specific inhibitor of HMGCS1, decreases cell growth both in AML cell lines and primary bone marrow cells of AML patients. Furthermore, combination of hymeglusin and the common chemotherapeutic drug cytarabine and adriamycin (ADR) had synergistic toxic effects on AML cells. Our study demonstrates the important role of HMGCS1 in AML, and targeting this protein is promising for the treatment of RR AML.

Combining the novel FLT3 and MERTK dual inhibitor MRX-2843 with venetoclax results in promising antileukemic activity against FLT3-ITD AML

FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately one third of acute myeloid leukemia (AML) patients. FLT3-Internal tandem duplication (FLT3-ITD) mutations are the most common FLT3 mutations and are associated with a poor prognosis. Gilteritinib is a FLT3 inhibitor that is US FDA approved for treating adult patients with relapsed/refractory AML and a FLT3 mutation. While gilteritinib monotherapy has improved patient outcome, few patients achieve durable responses. Combining gilteritinib with venetoclax (VEN) appears to make further improvements, though early results suggest that patients with prior exposure to VEN fair much worse than those without prior exposure. MRX-2843 is a promising inhibitor of FLT3 and MERTK. We recently demonstrated that MRX-2843 is equally potent as gilteritinib in FLT3-ITD AML cell lines in vitro and primary patient samples ex vivo. In this study, we investigated the combination of VEN and MRX-2843 against FLT3-ITD AML cells. We found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-mutated AML cell lines and primary patient samples. Importantly, we found that VEN synergistically enhances cell death induced by MRX-2843 in FLT3-ITD AML cells with acquired resistance to cytarabine (AraC) or VEN+AraC. VEN and MRX-2843 significantly reduce colony-forming capacity of FLT3-ITD primary AML cells. Mechanistic studies show that MRX-2843 decreases Mcl-1 and c-Myc protein levels via transcriptional regulation and combined MRX-2843 and VEN significantly decreases oxidative phosphorylation in FLT3-ITD AML cells. Our findings highlight a promising combination therapy against FLT3-ITD AML, supporting further in vitro and in vivo testing.

In silico predicted compound targeting the IQGAP1-GRD domain selectively inhibits growth of human acute myeloid leukemia

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting the IQGAP1-GRD domain, and conducted SAR of the ‘fittest hit’ to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, resulted in G2/M arrest, and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition, and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

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.

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.

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 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 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 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 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.

Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.