Patient-derived Acute Myeloid Leukemia Cells Research Articles

Hotspot DNA Methyltransferase 3A (DNMT3A) and Isocitrate Dehydrogenase 1 and 2 (IDH1/2) Mutations in Acute Myeloid Leukemia and Their Relevance as Targets for Immunotherapy.

DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7-23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide-HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy.

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.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

Serum Amyloid A1 (SAA1) Secreted By the Stromal Microenvironment Drives Malignant Clonal Proliferation in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p&amp;lt;0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

Protective Function of Human Skin Mesenchymal Niches for Acute Myeloid Leukemia-Initiating Stem Cells

Leukemia cutis (LC) or leukemic cell infiltration in skin is one of the common extramedullary manifestations in acute myeloid leukemia (AML) and signifies a poorer prognosis. However, the pathogenesis and maintenance of LC have been understudied, limiting our understanding of its impact on AML progression and relapse. We recently have shown that the AML cells infiltrated in the mouse skin during steady state and after chemotherapy were capable to regenerate AML after serial transplantation (Sandhow et al., J. Exp. Med, 2023). Further characterization of the skin mesenchymal stem cells (MSCs) suggests that skin MSCs could maintain AML-initiating stem cells (LSCs) and protect them from chemotherapy, even to a greater degree than their bone marrow (BM) counterparts. These data suggest the protective role of skin mesenchymal niche in AML LSCs during chemotherapy. However, it remains unexplored whether human skin MSCs exert the same functional impact on human AML cells. We here have explored the role of human skin MSCs in AML maintenance and survival during chemotherapy by RNA sequencing and in vitro co-culture of patient-derived AML cells with primary human skin MSCs in comparison with BM MSCs. We first by prospective characterization identified human skin MSCs with a phenotype of CD45-CD235a-CD31-CD44+CD146- from healthy donors. RNA sequencing and Q-PCR revealed expression of key hematopoietic stem cell (HSC) regulatory genes including THPO, SCF, CXCL12 and LAMA4 in human skin MSCs. Interestingly, while HSC maintenance genes such as THPO and KITLG were significantly upregulated in skin MSCs (p&amp;lt;0.0001 and p&amp;lt;0.05, respectively), HSC negative regulator SPP1 was downregulated (p=0.05), pointing to a possible better hematopoiesis-supportive function of skin MSCs. In line with this, our functional studies indicated that skin MSCs displayed a superior protective effect on human AML cell line THP-1 than their BM cell counterparts during cytarabine (Ara-C) treatment in the in vitro co-culture. There were more residual chemo-resistant AML cells, reflected in the higher number of CD36+ AML cells after treatment with Ara-C in cocultures with skin MSCs, compared to that with BM MSCs (p=0.0065). This result was confirmed by functional assays showing the increased number of cobblestone area-forming cells (p=0.0298) and colony-forming units (p=0.038) within the residual AML cells co-cultured with skin MSCs. To investigate the mechanisms involved in the protective function of human skin MSCs, we have performed RNA-sequencing of the human skin and BM MSCs before and after co-culture with AML cells. This has revealed potential unique molecular pathways between AML and skin MSCs. Furthermore, there were dramatically less altered genes in the skin MSCs compared to BM MSCs after 3 day-coculture with THP1 AML cells (221 vs 1761 altered genes with adjusted p values &amp;lt;0.05). These data indicate that skin MSCs are likely more resistant to AML-remodeling, which could in turn maintain AML cells in quiescence status, thereby protecting AML cells from Ara-C treatment. This speculation is currently under validation. In addition, gene set enrichment analysis showed upregulated genes associated with fatty acid metabolisms and ribosome in skin MSCs compared to BM MSCs, providing molecular evidence for a potential better metabolic support of skin MSCs for AML cells. Taken together, our preliminary data indicate an important role of human skin MSCs in supporting and protecting AML cells during Ara-C treatment. We are further investigating the underlying mechanisms.

A Novel CD64 CAR-T Therapy for the Treatment of Monocytic Acute Myeloid Leukemia

Introduction: Acute Myeloid Leukemia (AML) is a difficult to treat, life threatening malignancy with overall survival remaining extremely poor, approximately 15% at 5 years. Recent advances in therapeutic approaches for AML have significantly improved outcomes, most notably venetoclax and azacitidine which induces a remission in approximately 70% of patients. Despite this, the majority of patients ultimately relapse and succumb to their disease. We and others have previously shown that more monocytic (M5 by FAB classification) AML cells demonstrate increased resistance to venetoclax therapy and that relapse after venetoclax and azacitidine treatment shows a monocytic phenotype in approximately 30% of patients (Pei, et. al. Cancer Discovery, 2020). These patients experience decreased overall survival compared to patients with non-M5 AML. Furthermore, we have demonstrated that AML with a dominant monocytic phenotype can arise from a unique subclass of leukemia stem cells (LSCs) that we have termed monocytic-LSCs (m-LSCs) due to their immunophenotype, including expression of CD64. m-LSCs are highly resistant to venetoclax-based therapies (Pei, et. al., Cancer Discovery, 2023). As venetoclax is increasingly utilized in AML treatment regimens, it is essential to identify methods of targeting this m-LSC subtype to prevent and treat relapsed and refractory disease. Methods: To more effectively target monocytic AML, we have developed a novel chimeric antigen receptor (CAR) T cell therapy targeting CD64, an antigen commonly expressed in monocytic cells and strongly expressed in the m-LSC population. We generated two second-generation CAR constructs targeting CD64, one with CD28 and the other with 41BB costimulatory domains. We expressed these CARs in healthy donor human T cells via lentiviral transduction and tested their function in vitro and in vivo. Results: We demonstrate that CD64 CAR-T cells effectively treat monocytic AML using pre-clinical models. CD64 CAR-T cells eliminate CD64+ leukemia cell lines in vitro in a dose dependent manner with an effector to target (E:T) ratio of 1:4 and higher eliminating 100% of Molm14 leukemia cells at 18 hours compared to 0-10% in mock T-cell controls. We also show that CD64 CAR-T cells eliminate established Molm14 leukemia in vivo, with 100% of CAR-T treated mice surviving over 50 days with no evidence of leukemia versus less than 30 days survival with high tumor burden in mock treated mice (p&amp;lt;0.01, Figure 1). We find that CD64 CAR-T cells with CD28 verses 41BB costimulatory domains are similarly efficacious against monocytic AML, though we find more prolonged persistence of CD64-41BBz CAR-T cells in mice with long term leukemia free survival. Furthermore, we demonstrate that CD64 CAR-T cells kill CD64+ primary patient derived AML cells in ex vivo co-culture, resulting in a 60-80% reduction in viable AML cells at 18 hours across 1:1 to 1:16 E:T ratios compared to mock T cell controls. We further show that CD64 CAR-T cells target m-LSCs and reduce the engraftment potential of primary AML cells in mice with a nearly 9-fold reduction in leukemia burden in CAR-T treated versus control mice. Finally, preliminary experiments with AML patient derived xenograft models further support the in vivo efficacy of CD64 CAR-T cell treatment. Conclusions: Our data provide promising preclinical evidence for the efficacy of a novel CD64 CAR-T therapy in the treatment of monocytic AML. We propose that using this regimen to augment AML therapy is a potentially powerful means to eradicate m-LSCs, which are inherently resistant to venetoclax, and thereby increase remission duration. This concept serves as the basis for an upcoming clinical trial of CD64-directed CAR-T cells for CD64 expressing AML. Figure 1: CD64 CAR-T cells eliminate AML in in vivo murine leukemia models. A) NSG mice leukemia models of GFP expressing Molm14, a CD64+ human leukemia cell line, experience leukemia clearance within 3 days after treatment with CD64-28z CAR-T cells, as shown by decreased bioluminescent signal in images of CAR-T treated mice. Mock transduced human T-cells from the same human donor serve as a control. B) Survival curves demonstrate 100% survival of CAR-T treated mice over the experiment (p&amp;lt;0.01).

Emodin derivative E35 and its combination with autophagy inhibitor against acute myeloid leukemia cells in vitro and in vivo

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with poor prognosis and high recurrence rate. The discovery of more effective therapeutic strategies for AML plays a crucial role. The present work showed that E35, a novel derivative of emodin, significantly inhibited cell proliferation and induced autophagy and apoptosis in AML cells. Treatment with E35 markedly induced Beclin-1, LC3-II, cleaved Caspase-9 and PARP, and suppressed mitogen-activated protein kinase (MAPK) pathway. E35 exposure evoked autophagic activity prior to apoptosis induction, and autophagy inhibition by 3-methyladenine (3-MA) dramatically increased E35-induced apoptosis in both AML cell lines and patient-derived AML cells. Nevertheless, study on AML xenograft model showed that the combination E35 with 3-MA exhibited much more inhibitory effects on leukemia cell growth in vivo. No obvious adverse reactions occurred in the xenograft animals administered E35 alone or its cotreatment with 3-MA. These findings suggest that E35 could exert anti-leukemia effects, and that the combination of E35 and autophagy inhibitor might prove a more highly efficient strategy for AML treatment.

HSP90 Inhibitor PU-H71 in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.

The MLL-Menin Interaction is a Therapeutic Vulnerability in NUP98-rearranged AML.

Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin-MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL-Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors.

TAK-981, a SUMOylation inhibitor, suppresses AML growth immune-independently.

Acute myeloid leukemia (AML) generally has an unsatisfactory prognosis despite the recent introduction of new regimens, including targeted agents and antibodies. To find a new druggable pathway, we performed integrated bioinformatic pathway screening on large OHSU and MILE AML databases, discovered the SUMOylation pathway, and validated it independently with an external data set (totaling 2959 AML and 642 normal sample data). The clinical relevance of SUMOylation in AML was supported by its core gene expression which is correlated with patient survival, European LeukemiaNet 2017 risk classification, and AML-relevant mutations. TAK-981, a first-in-class SUMOylation inhibitor currently under clinical trials for solid tumors, showed antileukemic effects with apoptosis induction, cell-cycle arrest, and induction of differentiation marker expression in leukemic cells. It exhibited potent nanomolar activity, often stronger than that of cytarabine, which is part of the standard of care. TAK-981's utility was further demonstrated in invivo mouse and human leukemia models as well as patient-derived primary AML cells. Our results also indicate direct and cancer cell-inherent anti-AML effects by TAK-981, different from the type 1 interferon and immune-dependent mechanism in a previous solid tumor study. Overall, we provide a proof-of-concept for SUMOylation as a new targetable pathway in AML and propose TAK-981 as a promising direct anti-AML agent. Our data should prompt studies on optimal combination strategies and transitions to clinical trials in AML.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR01.

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Rational polypharmacological targeting of FLT3, JAK2, ABL, and ERK1 suppresses the adaptive resistance to FLT3 inhibitors in AML.

Despite significant advancements in developing selective FMS-like tyrosine kinase 3 (FLT3) inhibitors, resistance to treatment is common even on continued therapy. Acquisition of on-target mutations or adaptation to MAPK, JAK2, and ABL signaling pathways drive treatment failure and disease relapse. Although combinatorial targeting of all escape routes in preclinical models demonstrated its efficacy, the clinical application is challenging owing to drug-drug interaction and differing pharmacokinetics of the inhibitors. We reasoned that selective polypharmacological targeting could lead to a durable response with reduced toxicity. A cell-based screening was carried out to identify inhibitors targeting FLT3, RAS-MAPK, BCR-ABL, and JAK2 to target the adaptive resistance observed with FLT3 inhibitors. Here, we show that pluripotin is an equipotent inhibitor of FLT3, BCR-ABL, and JAK2 in addition to inhibiting Ras-GAP and extracellular signal-regulated kinase 1 (ERK1). Structural modeling studies revealed that pluripotin is a type II kinase inhibitor that selectively binds with inactive conformations of FLT3, ABL, and JAK2. Pluripotin showed potent inhibitory activity on both mouse and human cells expressing FLT3ITD, including clinically challenging resistant mutations of the gatekeeper residue, F691L. Likewise, pluripotin suppressed the adaptive resistance conferred by the activation of RAS-MAPK pathways, BCR-ABL, and JAK2 signaling. Treatment with pluripotin curbed the progression of acute myeloid leukemia (AML) in multiple invivo models including patient-derived primary AML cells in mouse xenotransplants. As a proof of concept, we demonstrate that targeted polypharmacological inhibition of key signaling nodes driving adaptive resistance can provide a durable response.

Abstract 1673: Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors

Abstract Molecular glue degraders (MGDs) of neo-substrates that function by hijacking the CRBN E3 ligase are clinically validated as therapeutics for the treatment of hematological malignancies. Novel MGDs of GSPT1, a translation termination factor, elicit dysregulated protein translation termination and subsequent induction of TP53-independent apoptosis in acute myeloid leukemia (AML) cells through activation of the integrated stress response. In addition, dysregulation of translation termination triggered by GSPT1 degradation appears to be cytotoxic specifically to MYC-driven cancers including solid tumors with translational addiction. Phase 1 clinical trials of three novel GSPT1 MGDs have been recently launched for the treatment of AML and/or MYC-driven solid tumors (NCT02848001, NCT04336982, NCT05546268, and NCT05144334). Herein, we present novel GSPT1 MGDs and their pre-clinical results against AML and solid tumors. Our current lead GSPT1 MGDs induced rapid, potent, and durable degradation of GSPT1, resulting in strong inhibition of tumor cell growth in a variety of cell lines of MYC-driven solid tumor and blood cancer (AML and acute lymphoblastic leukemia). Moreover, in ex-vivo assays using a panel of patient-derived primary AML cells, C-10386 was highly sensitive with EC50s at the nanomolar range. Furthermore, a related analogue (C-14207) showed excellent rodent PK profiles and demonstrated dose-dependent PKPD relationship and excellent efficacy in the NCI-H1155 NSCLC xenograft model. Finally, our current lead compounds showed safer profiles in various normal cells compared to other known GSPT1 degraders. Collectively, we have identified novel GSPT1 MGDs as promising development candidates for the treatment of AML and solid tumors. Citation Format: Min Sung Joo, JaeYung Lee, Eun-Jung Kim, Dong Hyuk Ki, Hunmi Choi, Joonwoo Nam, Jaewoo Park, Keon Wook Kang, Wooseok Han. Discovery of potent, selective, and orally bioavailable GSPT1 degraders and their pre-clinical anti-tumor activity in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1673.

RNA demethylase ALKBH5 promotes tumorigenesis of t (8;21) acute myeloid leukemia via ITPA m6A modification

BackgroundAlthough t (8;21) is in fact considered a good risk acute myeloid leukemia (AML), only 60% of the patients live beyond 5 years after diagnosis. Studies have shown that RNA demethylase ALKBH5 promotes leukemogenesis. However, the molecular mechanism and clinical significance of ALKBH5 in t (8;21) AML have not been elucidated.MethodsThe expression of ALKBH5 was assessed in t (8;21) AML patients via qRT-PCR and western blot. The proliferative activity of these cells was examined through CCK-8 or colony-forming assays, while flow cytometry approaches were used to examine apoptotic cell rates. The in vivo role of ALKBH5 promoting leukemogenesis was assessed using t (8;21) murine model, CDX, and PDX models. RNA sequencing, m6A RNA methylation assay, RNA immunoprecipitation, and luciferase reporter assay were used to explore the molecular mechanism of ALKBH5 in t (8;21) AML.ResultsALKBH5 is highly expressed in t (8;21) AML patients. Silencing ALKBH5 suppresses the proliferation and promotes the apoptosis of patient-derived AML cells and Kasumi-1 cells. With integrated transcriptome analysis and wet-lab confirmation, we found that ITPA is a functionally important target of ALKBH5. Mechanistically, ALKBH5 demethylates ITPA mRNA and increases its mRNA stability, leading to enhanced ITPA expression. Furthermore, transcription factor TCF15, specifically expressed in leukemia stem/initiating cells (LSCs/LICs), is responsible for the dysregulated expression of ALKBH5 in t (8;21) AML.ConclusionOur work uncovers a critical function for the TCF15/ALKBH5/ITPA axis and provides insights into the vital roles of m6A methylation in t (8;21) AML.

GSK3 inhibitor enhances gemtuzumab ozogamicin-induced apoptosis in primary human leukemia cells by overcoming multiple mechanisms of resistance.

In acute myeloid leukemia (AML), the heterogeneity of genetic and epigenetic characteristics makes treatment difficult. The prognosis for AML is therefore poor, and there is an urgent need for new treatments for this condition. Gemtuzumab ozogamicin (GO), the first antibody-drug conjugate (ADC), targets the CD33 antigen expressed in over 90% of AML cases. GO therefore has the potential to counter the heterogeneity of AML patients. However, a major clinical problem is that drug resistance to GO diminishes its effect over time. Here, we report that the inhibition of glycogen synthase kinase 3 (GSK3) alone overcomes several forms of GO resistance at concentrations without antileukemic effects. The GSK3 inhibitors tested significantly enhanced the cytotoxic effect of GO in AML cell lines. We elucidated four mechanisms of enhancement: (1) increased expression of CD33, the target antigen of GO; (2) activation of a lysosomal function essential for hydrolysis of the GO linker; (3) reduced expression of MDR1 that eliminates calicheamicin, the payload of GO; and (4) reduced expression of the anti-apoptotic factor Bcl-2. A similar combination effect was observed against patient-derived primary AML cells. Combining GO with GSK3 inhibitors may be efficacious in treating heterogeneous AML.

Heterogeneity of Patient-Derived Acute Myeloid Leukemia Cells Subjected to SYK In Vitro Inhibition

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a dismal prognosis. The cytoplasmic spleen tyrosine kinase (SYK) is highly expressed by hematopoietic cells and has emerged as a potential therapeutic target. In this study, we evaluated the in vitro antileukemic effects of five SYK inhibitors, fostamatinib, entospletinib, cerdulatinib, TAK-659, and RO9021, in a consecutive AML patient cohort. All inhibitors demonstrated a concentration-dependent antiproliferative effect, although there was considerable heterogeneity among patients. For fostamatinib and TAK-659, the antiproliferative effects were significantly higher in FLT3 mutated patients compared to nonmutated patients. Fostamatinib, entospletinib, TAK-659, and RO9021 induced significant apoptosis in primary AML cells, although the proapoptotic effects of the SYK inhibitors were less pronounced than the antiproliferative effects. Finally, most of the SYK inhibitors caused a significant decrease in the release of cytokines and chemokines from primary AML cells, indicating a potent inhibitory effect on the release of these leukemic signaling molecules. We concluded that the SYK inhibitors had antileukemic effects in AML, although larger studies are strongly needed to identify which patient subsets will benefit most from such a treatment.

Streptavidin-Drug Conjugates Streamline Identification of Optimal Toxic Payloads for Antibody-Based Hematopoietic Stem Cell Transplantation Conditioning

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) provides the best and possibly only chance for cure for acute myeloid leukemia (AML). In preparation for allo-HSCT, patients undergo conditioning with chemotherapy and/or irradiation to deplete host hematopoietic stem cells (HSCs), suppress the host immune system, and destroy residual leukemia cells. However, conditioning associated toxicities impose a formidable barrier to patients undergoing this potentially curative therapy. Antibody-drug conjugates (ADCs) have emerged as a promising and potentially safer alternative for allo-HSCT conditioning by allowing targeted depletion of the host HSC niche. Previously, we used saporin-conjugated CD45- or cKit-ADCs as the backbone of a novel conditioning regimen which enabled engraftment between fully mismatched mice. However, saporin ADCs were nonmyeloablative and showed modest antitumor activity. We hypothesized that fully myeloablative ADCs capable of enhanced antileukemia activity could be produced using alternative toxic payloads to saporin. However, direct conjugation of multiple candidate payloads and/or antibodies is costly, time-consuming, and laborious. Here, we describe the production of a panel of streptavidin (SAv)-drug conjugates which allows efficient ADC generation from any biotinylated protein. This flexible platform enabled the rapid production of CD45-ADCs conjugated to multiple toxic payloads, facilitating in vitro screening of their ability to target HSCs and leukemia cells. METHODS: Azide-alkyne cycloaddition ("Click" chemistry) was used to covalently link SAv-azide (Protein Mods, Inc) with dibenzocyloocytne (DBCO)-coupled drug payloads (Levena Biopharma) (Fig. 1). We conjugated three toxic payloads to SAv for these studies: monomethyl auristatin E (MMAE), Duocarmycin SA (Duo), and PNU-159682 (PNU). All of these payloads contain cleavable valine-citrulline linkers for payload release following ADC internalization. For the conjugation reactions, SAv-azide was combined with DBCO-coupled payload and incubated 2 hours at 20oC, with 20% DMSO added as cosolvent. Free payload and DMSO were removed using size exclusion chromatography and dialysis. ADCs were freshly prepared before use by mixing SAv-drug conjugates in a 1:1 molar ratio for 15 minutes with biotinylated antibodies. For our initial studies, we evaluated murine anti-CD45.2 (Clone 104) and human anti-CD45 (Clones BC8 and HI30). Non-biotinylated antibodies combined with SAv-drug conjugates were used as negative controls. All ADCs were tested for cytotoxicity in vitro against 1) murine YAC-1 (mCD45.2+) and human Jurkat (mCD45.2-) cell lines using XTT viability assays, 2) primary HSCs from B6 mice using methylcellulose assays, and 3) AML1 primary murine leukemia cells (Dnmt3aR878H/+ and FLT3-ITD; provided by Dr. Tim Ley) using methylcellulose assays. IC50 values for each assay were derived with nonlinear regression of dose response data. RESULTS: Mouse CD45-Duo and CD45-PNU were highly effective at killing AML1 cells (IC50 values: 39.2pM and 61.1pM, respectively; Fig. 2), as well as B6 HSCs (IC50 values: 0.85nM and 0.13nM, respectively) in vitro. Notably, mouse CD45-MMAE was ineffective against YAC-1 cells, B6 HSCs, and AML1 cells, suggesting that MMAE is not an effective payload for ADC-based conditioning in the mouse. Importantly, nonbiotinylated antibodies mixed with SAv-drug conjugates were largely ineffective, indicating the need for the antibody to bind the SAv-drug conjugate to achieve specific toxicity. Preliminary studies using human CD45-MMAE and CD45-Duo demonstrated that while both payloads could yield ADCs able to kill Jurkat cells, ADCs made using human antibody clone BC8 were considerably more effective than ADCs made with clone HI30. CONCLUSIONS: Our novel SAv-drug conjugate successfully enabled rapid generation of ADCs with a number of potential toxic payloads. This system identified multiple effective candidate payloads for use in mouse and human ADCs for targeting HSCs and leukemia cells in vitro and in vivo. Our ongoing and future studies with this system are focused in three areas: 1) production and testing of mouse and human cKit ADCs, 2) using mouse ADCs to condition for murine HSCT and target AML1 cells in vivo, and 3) evaluating killing of human cord blood-derived HSCs and patient-derived AML cells with human ADCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acute Myeloid Leukemia Cells Rely on the Glycolytic Enzyme PGK1 to Support Energy Production and Amino Acid Metabolism

Acute myeloid leukemia (AML) cells frequently display an increased dependence on glucose compared to healthy hematopoietic stem and progenitors (HSPCs). Furthermore, increased glucose metabolism is associated with worse outcomes in AML. However, the upstream regulators that drive glucose metabolism as well as how glucose is utilized in AML remains largely unknown. Here, we have discovered that the oncogenic transcription factor (TF), JUN supports AML, in part, by regulating glycolytic enzymes that utilize glucose to support both cellular energetics and amino acid metabolism. Having previously reported that certain aggressive AML sub-types depend on JUN, we next set out to identify JUN-regulated transcriptional programs that support AML. To do so, we engineered the human AML cell line, THP-1 to express either control (shNT) or JUN-targeting shRNAs and then performed RNA-seq on cells from each condition. Next, we carried out a functional enrichment analyses and discovered that genes encoding numerous enzymatic regulators of glycolysis were significantly down-regulated by JUN-inhibition. Using qPCR validation, we confirmed that JUN inhibition results in reduced mRNA expression of Hexokinase 1 & 2 (HK1 & HK2), Glucose-Phosphate Isomerase (GPI), Phosphofructose Kinase (PFKP), Aldolase A (ALDOA), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), Phosphoglycerate Kinase (PGK1), Enolase 1 (ENO1) and Pyruvate Kinase Muscle (PKM). ChIP assays confirmed that JUN directly localizes to the promoters of each of these genes except for ALDOA and PKM. To assess whether the JUN-dependent changes in glycolytic gene expression translated to defects in the ability of AML cells to catabolize glucose, we performed a glycolysis stress test on shNT- and shJUN-expressing human AML cell lines (e.g. THP-1, NOMO-1 and MOLM-14). From this assessment, we found that JUN inhibition resulted in a complete loss of glucose catabolism in all cell lines tested. We postulated that JUN activates glycolytic gene expression to support AML and thus, inhibition of JUN-regulated glycolytic genes will impede leukemia cell function. To test this hypothesis, we generated shRNAs targeting each of the JUN-regulated glycolytic enzymes. We then engineered mouse leukemia cells expressing the human leukemogenic allele, MLL-AF9 - found in both childhood and adult AML - to express these shRNAs and then assessed their colony forming capacity (CFC) in cytokine-enriched semisolid culture. In parallel, we also assessed how knockdown of each glycolytic enzyme impacted the CFC of healthy HSPCs. Notably, we observed that inhibition of each enzyme significantly impaired the CFC of MLL-AF9 leukemia cells but that inhibition of Pfkp, Aldoa or Pgk1 did not impact the CFC of healthy HSPC, suggesting that these enzymes were selectively important for AML. We next evaluated how knockdown of Pfkp, Aldoa or Pgk1 impacted leukemia propagation in vivo. Interestingly, inhibition of Pfkp or Pgk1, but not Aldoa, significantly delayed the onset of MLL-AF9 driven leukemia in vivo. Moreover, we found that a chemical inhibitor of PGK1, CBR-470-1, reduces the CFC of patient-derived AML cells in vitro. Since Pgk1 had not previously been studied in AML, we focused our attention on the its role in leukemia cell metabolism. We thus performed untargeted metabolomics of shNT- and shPgk1-expressing mouse MLL-AF9 cells using hydrophilic liquid chromatography mass spectrometry (HILIC-MS). PGK1 utilizes ADP to convert 1,3-bisphosphoglycerate into 3-phosphoglycerate (3-PG) and ATP. 3-PG can subsequently be used to generate phosphoenolpyruvate (PEP) or in the de novo synthesis of serine (Ser). As expected, we found that Pgk1 inhibition resulted in a significant decrease in 3-PG, PEP and Ser. We also observed that Pgk1 inhibition resulted in the decrease of several tricarboxylic acid cycle (TCA) intermediates that support oxidative phosphorylation energy production. Surprisingly, we also found that Pgk1 inhibition resulted in a marked decrease in several amino acids, including Alanine, Arginine and Histidine. We are currently investigating whether the decrease in these amino acids mediated by Pgk1 inhibition is due to an impediment of their biosynthesis or their consumption in gluconeogenic pathways. Collectively, our studies identify JUN as a previously unrecognized regulator of AML cell metabolism and shed new into how glucose is catabolized in AML cells.

VIP943 Is a Novel CD123 Antibody Drug Conjugate with in Vitro and In Vivo Efficacy in Acute Myeloid Leukemia (AML) Models

Background: Current treatment options (cytarabine with anthracyclines) for patients with acute myeloid leukemia (AML) are often associated with severe and barely tolerable toxicities. In patients who tolerate induction therapy, the complete remission rate is 60%-80%; however, 50% of patients relapse due to the presence of leukemic stem cells (LSC). Antibody-drug conjugates (ADCs) are putatively designed to deliver potent drugs directly to cancer cells while sparing normal tissue; however, first-generation ADCs are associated with severe bone marrow and liver toxicity. VIP943 is a CD123-targeting ADC which uses a kinesin spindle protein inhibitor (KSPi) as a new payload class and a unique legumain linker designed to overcome liabilities of currently approved ADC therapies. The payload is delivered with a drug-antibody ratio of 6. It consists of the KSPi warhead which is modified with a CellTrapperTM moiety so it accumulates within the CD123-targeted tumor cell and cannot enter normal cells. Non-human primate toxicology studies demonstrated a highly favorable and differentiated safety profile for VIP943 (Kirchhoff et al, Cancers 2020). Here we characterize VIP943 in various patient-derived AML models. Methods: For co-localization experiments, MOLM-13 AML cell line was stained with labelled anti-CD123 antibody or isotype control and CytoPainter LysoGreen indicator reagent and analyzed by imaging flow cytometry. AML primary patient mononuclear cells from blood and bone marrow were treated with VIP943 for 24, 48, and 72 hours. Flow cytometry immunophenotyping for AML blasts and CD34+/CD38- LSCs was used to analyze these populations. An in vivo patient-derived AML PDX mouse model was treated with VIP943 (5 mg/kg IV every 7 days) or in combination with 5-azacytidine (2.5 mg/kg SC days 1-5 x 3) and venetoclax (50 mg/kg PO days 1-5 x 3). Cytokine release syndrome was analyzed comparing VIP943 to isotype control ADC, anti-CD123 antibody, and positive comparator antibodies by in vitro cytokine release assays (CRA) in two different assay formats using whole blood and isolated mononuclear cells from 10 healthy donors. Flow cytometric lymphocyte and leukemic immunophenotyping was performed from blood of non-human primates. A leukemia cell line panel drug screen with VIP943 was evaluated and baseline genomic features were correlated with response. Results: Co-localization studies confirm specific internalization and efficient intracellular trafficking to the lysosome of VIP943. In vitro treatment with VIP943 (66 nM, continuous exposure) delivers blast reductions of 37% after a 24h incubation with VIP943, 45% after 48h incubation and 83% after an 72h incubation. In the CD34+/CD38- cell population, a reduction of 71% was achieved. In a patient-derived AML model, VIP943 with 5-azacytidine and venetoclax led to complete tumor growth inhibition after 21 days showing superiority of this triplet therapy (complete regression: 8/9 mice) with high tolerability (maximum body weight loss -4.8 %). The immunotoxicological evaluation in both CRA formats and with the tested antibody concentrations (100µg, 10µg, 1µg) exerts no induction of cytokines by VIP943 compared to controls. Immunophenotyping demonstrates no impact of VIP943 treatment on lymphocytic subpopulations, whereas the CD123-positive basophils are reduced after 24h followed by an almost complete recovery. VIP943 IC50 ranges from 0.1nM-300nM in a leukemia cell line panel screen and features associated with sensitivity are investigated. Conclusions: VIP943 is a next-generation ADC with a differentiated safety profile from currently approved ADCs, including lack of in vitro cytokine release. In vitro and in vivo studies using patient-derived AML cells show VIP943 has favorable monotherapy and combination efficacy including targeting of leukemic stem cells, which drive relapse. These findings warrant evaluating VIP943 in clinical trials.