Preclinical Models Of Acute Myeloid Leukemia Research Articles

The multi-CDK inhibitor dinaciclib reverses bromo- and extra-terminal domain (BET) inhibitor resistance in acute myeloid leukemia via inhibition of Wnt/β-catenin signaling

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions, regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development, but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML, both as monotherapy and in combination. To date, many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9, which has a known physical and functional interaction with the BET protein BRD4. Therefore, we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here, we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines, primary AML samples, and in vivo. Further, we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway, a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels, including downregulation of β-catenin, the Wnt co-receptor LRP6, as well as many Wnt pathway components and targets. Moreover, dinaciclib sensitivity remains unaffected in a setting of BET resistance, demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately, our results demonstrate rationale for combination CDKi and BETi in AML. In addition, our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML.

Mezigdomide is effective alone and in combination with menin inhibition in preclinical models of KMT2A-r and NPM1c AML

AbstractSmall molecules that target the menin-KMT2A protein-protein interaction (menin inhibitors) have recently entered clinical trials in lysine methyltransferase 2A (KMT2A or MLL1)–rearranged (KMT2A-r) and nucleophosmin-mutant (NPM1c) acute myeloid leukemia (AML) and are demonstrating encouraging results. However, rationally chosen combination therapy is needed to improve responses and prevent resistance. We have previously identified IKZF1/IKAROS as a target in KMT2A-r AML and shown in preclinical models that IKAROS protein degradation with lenalidomide or iberdomide has modest single-agent activity yet can synergize with menin inhibitors. Recently, the novel IKAROS degrader mezigdomide was developed with greatly enhanced IKAROS protein degradation. In this study, we show that mezigdomide has increased preclinical activity in vitro as a single-agent in KMT2A-r and NPM1c AML cell lines, including sensitivity in cell lines resistant to lenalidomide and iberdomide. Further, we demonstrate that mezigdomide has the greatest capacity to synergize with and induce apoptosis in combination with menin inhibitors, including in MEN1 mutant models. We show that the superior activity of mezigdomide compared with lenalidomide or iberdomide is due to its increased depth, rate, and duration of IKAROS protein degradation. Single-agent mezigdomide was efficacious in 5 patient-derived xenograft models of KMT2A-r and 1 NPM1c AML. The combination of mezigdomide with the menin inhibitor VTP-50469 increased survival and prevented and overcame MEN1 mutations that mediate resistance in patients receiving menin inhibitor monotherapy. These results support prioritization of mezigdomide for early phase clinical trials in KMT2A-r and NPM1c AML, either as a single agent or in combination with menin inhibitors.

Enhancing anti-AML activity of venetoclax by isoflavone ME-344 through suppression of OXPHOS and/or purine biosynthesis in vitro

Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 enhanced VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, a purine biosynthesis inhibitor, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired AraC resistance showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. In vivo studies revealed significantly prolonged survival upon combination therapy of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia compared to the vehicle control. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.

Abstract A013: Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286

Abstract BAF chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer as well as other diseases. FHD-286 is a first-in-class dual inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4) that is being developed for the treatment of advanced hematologic malignancies. We previously reported that exposure of acute myeloid leukemia (AML) cells to pharmacologically relevant concentrations of FHD-286 led to downregulation of leukemic stem cell (LSC) marker CD34, and upregulation of the myeloid maturation marker CD11b. Morphologic features of differentiation were also observed, suggesting that BAF functions to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by reducing their capacity for stem cell renewal. Using single-cell transcriptomic data, we further assessed the impact of FHD-286 on differentiation markers of tumor cells from clinical AML patients. scRNA-seq data was obtained for 26 bone marrow aspirate samples from 11 AML and 2 myelodysplastic syndrome (MDS) patients from the Phase 1 clinical study evaluating FHD-286. Non-tumor cell types (T-cells, erythroid, B cells, megakaryocytes) were identified using published gene markers and removed from the dataset, leaving only tumor cells enriched for either LSC markers (CD34, CD123) or CD11b. We next identified differentially expressed genes between patient LSCs and CD11b+ cells. The top 50 genes associated with each cell group were taken as signature markers of stemness or differentiation, and their mean expression used as phenotypic scores for all patient tumor cells. Clustering analysis of the patient single-cell data showed that cells grouped according to cell type, treatment timepoint, and patient identity. Tumor cells expressing LSC markers CD34 and CD123 clustered separately from those expressing differentiation marker CD11b, and the relative percentage of CD11b+ tumor cells increased on treatment for 8 out of 12 patients dosed at >=5mg, consistent with preclinical AML models. Differential expression analysis revealed additional genes upregulated in either LSCs or CD11b+ cells, which were identified in literature as markers of stemness or myeloid differentiation, respectively. Single-cell LSC and differentiation signature scores showed a strongly statistically significant shift away from LSC identity and toward differentiation for 10 out of 12 patients. Single-cell RNA data from AML patients validates the preclinical observation that FHD-286 treatment induces changes in LSCs resembling differentiation towards a terminal myeloid cell state. This dataset further indicates that nearly all patients undergo some degree of tumor cell differentiation after treatment, suggesting broad on-target activity of FHD-286. Citation Format: GiNell Elliott, Jessica Wan, Kim Horrigan, Mike Collins, Astrid Thomsen, Ashley K Gartin, Ammar Adam, David L Lahr, Gabriel Sandoval, Sameul Agresta, Jessica Piel, Martin Hentemann. Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A013.

Efficacy of Belvarafenib with and without Cobimetinib in Preclinical Models of Ras Pathway-Mutant AML

Introduction Outcomes for children with acute myeloid leukemia (AML) remain poor, with ~40% dying from refractory leukemia or treatment-related toxicity (Gamis et al. 2014). NRAS, KRAS, and NF1 mutations occur in over 40% of pediatric AMLs (Bolouri et al. 2018), but efforts to therapeutically target the RAS/mitogen activated protein kinase (MAPK) pathway have been largely unsuccessful. Belvarafenib, a novel type II pan-RAF kinase inhibitor that inhibits mutant monomeric BRAF proteins and activated RAF homo- and heterodimers, has shown promising safety/efficacy data in adult solid cancers (Yen et al. 2021). Here, we investigate belvarafenib in preclinical models of AML, both alone and in combination with the allosteric MEK inhibitor, cobimetinib. Methods We used a panel of NRAS (OCI-AML3, HL-60, THP-1) or KRAS (NOMO-1, NB4, and SKM-1) mutant human AML cell lines. Viability was determined by CellTiter-Glo. Synergy was assessed by Bliss Independence and Chou Talalay methods. Transcriptome and proteomic profiling were performed as previously described (Pucciarelli et al. 2020). Mouse AMLs were generated using retroviral insertional mutagenesis (Li et al. 2011). Cryopreserved primary AML cells were injected intravenously into sublethally irradiated recipients that were then treated daily with vehicle, belvarafenib, cobimetinib, or the combination until disease progression. Survival curves were generated using Kaplan-Meier analysis. Results Belvarafenib inhibited the growth of AML cells lines with nanomolar potency at IC50 values ranging from 48nM (OCI-AML3) to 310nM (SKM-1). In all cell lines, belvarafenib and cobimetinib were highly synergistic. Western blotting of OCI-AML3 cells treated with either belvarafenib or cobimetinib at their respective IC50 values (50nM belvarafenib, 20nM cobimetinib) for 4 or 24 hours demonstrated discordant effects on downstream MAPK effector proteins (Fig. 1). Whereas cobimetinib potently suppressed phosphorylated ERK (pERK), belvarafenib unexpectedly had no effect. Phosphorylated S6 (pS6) levels were not reduced by either belvarafenib or cobimetinib at their respective IC50 values, but were dramatically reduced by the drug combination. To further investigate these unanticipated biochemical findings, we treated OCI-AML3 cells with belvarafenib, cobimetinib or the combination and performed kinome profiling and transcriptome sequencing (RNAseq) analysis. Exposure to 10nM cobimetinib inhibited several kinases at 4 hours with rebound activation of kinases involved in cell cycle progression at 24 hours. Cells treated with 100nM belvarafenib displayed down-regulation of multiple kinases that persisted at 24 hours. Cells treated with the combination at these doses exhibited the most potent inhibition of many kinases; this effect recapitulated the effect of high dose (500nM) belvarafenib, but not high dose (50nM) cobimetinib. At the transcriptional level, cells treated with the combination showed far greater suppression of negative regulators of MAPK signaling (e.g., DUSPs, SPRY2/4, SPRED1/2) than either agent alone, suggesting potent inhibition of the MAPK pathway by the combination. Intriguingly, OCI-AML3 cells treated with high-dose belvarafenib or low-dose combination unexpectedly exhibited profound down-regulation of mTORC1-regulated genes on GSEA analysis. We extended these in vitro data by performing preclinical trials in mice transplanted with 5 independent primary Nras- or Kras-mutant AMLs that received belvarafenib (15mg/kg), cobimetinib (2mg/kg), the combination or vehicle by oral gavage (Fig. 2). The combination was well tolerated. Belvarafenib prolonged survival in all 5 trials; in 3 of 5, the addition of low-dose cobimetinib further enhanced survival (median survival: vehicle, 9 days; cobimetinib, 13 days; belvarafenib, 22 days; combination, 32 days; p < 0.0001). Conclusions Belvarafenib showed activity in 6 of 6 Ras mutant human AML cell lines and in 5 of 5 primary Ras mutant murine AMLs. Belvarafenib and cobimetinib displayed synergy in all AML cell lines and in 3 of 5 murine AMLs treated in vivo. Mechanistically, we identified distinct biochemical and transcriptional effects of RAF dimer and MEK inhibition in AML cells. We are characterizing these further and pursuing causes of resistance in primary Nras- and Kras-mutant mouse AMLs that relapsed after an initial response to treatment.

REM-422, a Potent, Selective, Oral Small Molecule mRNA Degrader of the MYB Oncogene, Demonstrates Anti-Tumor Activity in Mouse Xenograft Models of AML

The c-MYB (MYB) oncogenic transcription factor is a key regulator of hematopoietic cell differentiation and proliferation. Recurrent genetic lesions or dysregulation of MYB have been identified in a variety of cancers, including adenoid cystic carcinoma (ACC), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), pediatric low-grade glioma (pLGG), and others. AML is characterized by multiple oncogenic abnormalities and high MYB mRNA expression. Evidence from in vitro and in vivo animal studies suggests that MYB may be a key downstream effector of these oncogenic abnormalities through the direct regulation of genes such as BCL2, MYC and FLT3. Functional genomics studies reveal MYB dependencies in human leukemia cell lines and mouse models of leukemia. Together, these data suggest that MYB is an attractive therapeutic target for AML and other hematological malignancies. Therapeutic agents that directly target MYB have yet to be developed, presumably due to the challenges of targeting this intrinsically disordered transcription factor, which lacks tractable ligand binding pockets. Here, we describe the mechanism of action and biological activity of REM-422, a potent, selective, oral small molecule messenger RNA (mRNA) degrader of MYB. REM-422 was identified through screening and medicinal chemistry efforts directed towards identifying small molecules that induce the inclusion of a normally unused ‘poison exon’ (PE), which contains a premature termination codon, into the MYB pre-mRNA transcript. Poison exon inclusion activates the nonsense-mediated decay pathway and promotes the degradation of MYB mRNA. We demonstrate in a cell-free assay that REM-422 acts by selectively facilitating the binding of the U1 snRNP spliceosome complex, which governs exon usage by recognizing 5‘ splice sites, to oligonucleotides containing the MYB PE 5‘ splice site sequence. REM-422 treatment of human leukemia cell lines promotes poison exon inclusion, leading to degradation of MYB mRNA and subsequent reduction in MYB protein levels (Figure 1A). In a panel of AML cell lines, REM-422 demonstrated preferential anti-proliferative activity in cell lines with high MYB expression, and in those shown to be MYB-dependent through functional genomics studies. To understand the pharmacokinetic (PK) and pharmacodynamic (PD) effects of MYB in vivo, Kasumi-1 AML cells were implanted subcutaneously in CB17 scid immunocompromised mice and REM-422 was dosed by oral gavage after the formation of established tumors. QD PO administration of REM-422 demonstrated dose-dependent effects in tumors on MYB mRNA levels, with induction of the MYB poison exon leading to reductions of MYB functional mRNA. In a 21-day study, REM-422 demonstrated significant anti-tumor activity, including tumor regressions at well tolerated dose levels (Figure 1 left). To further investigate the activity of REM-422, AML patient cells were injected into NOG immunocompromised mice and allowed to engraft. After successful engraftment, mice were dosed by oral gavage once daily with REM-422 at 10 mg/kg or vehicle for 24 days. Flow cytometry analysis of bone marrow and peripheral blood samples demonstrated that REM-422 led to the eradication of human leukemia cells from both compartments (Figure 1 right). Together, these data demonstrate that REM-422 treatment leads to degradation of MYB mRNA in preclinical models of AML and supports the therapeutic potential for REM-422 in AML patients.

The PP2A-B56α Heterocomplex Regulates Response to Venetoclax Plus Azacitidine Treatment in AML

Venetoclax (VEN), a selective inhibitor of BCL2, combined with azacitidine (Aza) has emerged as the new standard of care for patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. However, the long-term medical benefit is modest, and relapse appears to be unavoidable. We have recently reported that PP2A activators enhance the efficacy of VEN and VEN+Aza in primary samples and pre-clinical models of AML, and that the PP2A-B56α complex drives the synergistic pro-apoptotic activity observed. Our aim here is to further decipher the role of PP2A-B56α in VEN+Aza treatment response in AML. We used CRISPR-Cas9 to generate HL-60 sublines lacking the B56α or B55α PP2A regulatory subunits. Interestingly, while there were no significant differences in their baseline rate of proliferation, HL-60 B56α knockdown (KD) clones were significantly more resistant to VEN and VEN+Aza than wild type (WT) cells. In contrast, HL-60 B55α KD clones were sensitive to VEN+Aza, pointing out a specific role for specific PP2A heterotrimers, in this case the PP2A-B56α complex in the combined treatment response. Protein expression analysis of known substrates of PP2A-B56α demonstrated that pSer62-MYC and total MYC decreased after VEN+Aza treatment in WT cells but not in KD cells. It is known that the PP2A-B56α complex directly dephosphorylates MYC-Ser62, inducing MYC ubiquitin-mediated proteasomal degradation. Moreover, p21/CDKN1A, that is transcriptionally activated by p53 and inhibited by MYC, was up-regulated at both the mRNA and protein level after VEN+Aza treatment in WT cells but not in KD cells. HL-60 is a p53 null cell line; therefore, p21 up-regulation upon the combined treatment might be MYC-dependent. Of note, treatment of HL-60 B56α KD cells with the MYC inhibitor MYCi975 rescued the VEN+Aza synergy seen in these cells, confirming the relevance of MYC degradation to VEN+Aza response. Interestingly, VEN+MYCi975 had synergistic anti-leukemic effects in these same cell lines. Furthermore, CDKN1A expression was also rescued upon the triple therapy consisting of VEN+Aza+MYCi975. Combined, our results suggest that the PP2A-B56α complex is involved in the regulation of VEN+Aza response through changes in MYC expression. Interestingly, a novel small molecular glue (PMG) developed by our group, which specifically stabilizes the PP2A-B56α complex, enhanced VEN plus Aza response in vitro; however, this triple therapy did not work in HL-60 B56α KD cells and we observed a clearly decrease in MYC protein expression upon the triple therapy only in HL-60 B56α WT cells, supporting the rationale to translate these novel PMGs into the clinic. Altogether, our data suggest that PP2A-B56α might have an important role in VEN plus Aza treatment response through the regulation of MYC degradation.

Restoration of LAT Signaling in CD33-Directed CAR T Cells Improves Potency and Persistence Against Acute Myeloid Leukemia

Despite the development of novel therapies, the majority of patients with acute myeloid leukemia (AML) will not achieve long-term remissions. Patients with relapsed/refractory (r/r) AML have particularly poor outcomes, as conventional chemotherapy regimens are often unable to elicit long-term remissions. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative in eligible patients, however, bridging therapies which can induce deep remissions prior to HSCT are needed. Second generation chimeric antigen receptor (CAR) T cells targeting lineage-restricted antigens, such as CD19, have induced remissions in up to 80 - 90% of pediatric patients with r/r B-lineage acute lymphoblastic leukemia (ALL), however, post-CAR relapses are common and only ~50% of treated patients achieve durable remissions. Similar strategies targeting myeloid-restricted antigens with 2 nd generation CAR T cells in AML have not replicated the success seen in ALL. As clinical experience has highlighted the impact of CAR T cell potency, persistence, and antigen-sensitivity to long-term efficacy in ALL, strategies to boost these properties in myeloid-directed CAR T cells are necessary to extend the clinical benefit of this therapy to patients with AML. Utilizing a global phosphoproteomics screen, we recently identified that inefficient phosphorylation of the Linker for activation of T cells (LAT) and the subsequent decrease in the downstream signaling pathways results in suboptimal CAR T cell activation. To overcome inefficient LAT activation, we developed a novel bicistronic CAR platform, consisting of a 2 nd generation CAR along with an Adjunctive LAT-Activating CAR (ALA-CART) which improved sensitivity to low-antigen leukemia cells, increased in vivo potency, and enhanced persistence in xenograft models of ALL. We hypothesized that the restoration of LAT signaling via the ALA-CART platform would also improve potency and persistence in pre-clinical models of AML. We found that while CD33 ALA-CART cells responded to CD33+ AML cells in vitro, they were less cytotoxic and produced lower levels of cytokines than CD33-28z or CD33-BBz 2 ndgeneration CAR T cells. However, CD33 ALA-CART cells demonstrated increased potency in xenograft models, eradicating AML cells at much lower doses than CD33-28z and CD33-BBz CAR T cells (Fig. 1A, p<0.0001). This in vivo potency correlated with an enrichment of T stem cell memory cells in the ALA-CART product, resulting in increased persistence of CD33 ALA-CART cells after leukemia clearance (Fig. 1B, p=0.0139). The ALA-CART platform was associated with increased mitochondrial mass and function, suggesting that the increased signaling through LAT results in an enhanced capacity for oxidative phosphorylation, potentially contributing to the increased persistence of CD33 ALA-CART cells in vivo. Thus, the ALA-CART platform, designed to restore LAT signaling in CAR T cells, demonstrates that CAR molecules can be rationally designed to enhance both in vivo potency and persistence, and represents a promising strategy for the development for more effective AML-directed CAR T cells.

The Combination of Lanraplenib, a Selective SYK Inhibitor, and Gilteritinib, a FLT3 Inhibitor, Targets Aberrant Proliferation and Differentiation Blockade in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by uncontrolled clonal proliferation of poorly differentiated myeloid progenitor cells in the bone marrow and blood. Deregulated spleen tyrosine kinase (SYK) impairs myeloid differentiation and has been implicated in the pathogenesis of AML driven by FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation (FLT3-ITD). While SYK is rarely mutated, it is highly activated in FLT3-ITD-mutated AML and directly phosphorylates FLT3-ITD, resulting in aberrant expression of multiple oncogenic pathways. Lanraplenib (LANRA) is a selective, next-generation, SYK inhibitor that has shown favorable PK, PD, and safety in healthy volunteers and patients with autoimmune diseases. LANRA is currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). By jointly inhibiting the leukemogenic network created by SYK and mutant FLT3, LANRA and gilteritinib potentially provide a new treatment regimen in a patient population with few treatment options. Method: AML cell viability in response to LANRA, gilteritinib or their combination was assessed by CellTiter Glo (CTG) at 5 days. Apoptosis was evaluated by measuring Annexin V and propidium iodide staining by flow cytometry. Myeloid differentiation was assessed by flow cytometry using CD11b and CD14 cell surface markers and/or immunohistochemistry. In addition, RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. In vivo studies were conducted using the MV4;11 cell line-derived xenograft model. LANRA was administered twice daily at a dose of 75 mg/kg via subcutaneous injections, either alone or in combination with gilteritinib at a dose of 2 mg/kg dosed once daily by oral gavage, for 28 days. Results: Treatment of FLT3-ITD mutated AML cells with LANRA results in dose- and time-dependent increase in myeloid differentiation and reductions in leukemic cell viability. RNA-seq analysis of MV4;11 cells revealed that LANRA abrogates multiple downstream FLT3-ITD leukemogenic signaling pathways including JAK/STAT3/5, MAPK, mTOR and OXPHOS. Combined pharmacologic inhibition of SYK with LANRA and FLT3-ITD with gilteritinib results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Additionally, the combination of LANRA and gilteritinib demonstrated increased differentiation and apoptosis induction in vitro. LANRA in combination with gilteritinib showed significant tumor growth inhibition compared to either single agent in a subcutaneous FLT3-ITD AML cell line-derived xenograft mouse model. In line with these observations, a retrospective analysis of bone marrow-engrafted AML cells from a disseminated FLT3-ITD AML patient-derived xenograft mouse model showed evidence of increased myeloid differentiation and inhibition of cell proliferation. Conclusion: In preclinical models, LANRA demonstrates compelling anti-leukemic activity in combination with gilteritinib. By jointly targeting hyperproliferation and differentiation blockade, LANRA and gilteritinib may provide a new therapeutic approach that can be exploited in AML. Additionally, LANRA disrupts downstream signaling of key pathways often associated with resistance to gilteritinib, providing additional mechanistic support for continued testing of LANRA in combination with gilteritinib in FLT3-ITD-mutated AML patients. These studies helped motivate the ongoing clinical combination with gilteritinib in patients with (R/R) FLT3-mutated AML (NCT05028751).

Calibrated Release IL15 Bivalent CD33 and/or FLT3 and NOT Emcn Logic Gated Gene Circuit CAR-NK Cell Therapy (SENTI-202) in Venetoclax Resistant Patient Derived Xenograft Acute Myeloid Leukemia Models

Venetoclax (VEN) as combination therapy has improved response rates and overall survival of patients with acute myeloid leukemia (AML). However, once AML relapse prognosis is dire with a 5.3 month median survival (Brandwein, 2020). While chimeric antigen receptor (CAR) cell therapies have revolutionized the treatment landscape for lymphoid hematological malignancies, development of such therapies for AML has been challenging, in part due to the heterogeneity of the disease. Currently, there is a paucity of individual AML targets that are consistently expressed across AML subpopulations. Furthermore, the expression of these AML targets is not restricted to tumor cell populations, often resulting in off-tumor toxicity against healthy cell populations. SENTI-202 employs OR and NOT Logic Gating to overcome these challenges. SENTI-202 represents an innovative preclinical CAR-NK cell therapy, engineered to exploit a powerful CD33 OR FLT3 NOT EMCN Logic Gate gene circuit, in conjunction with calibrated release IL-15 (crIL15) expression. The CD33 OR FLT3 (OR GATE) activating CAR (aCAR) concurrently targets two AML antigens, thus permitting a broader therapeutic window against AML LSCs (CD33+/-, FLT3+), blasts (CD33+, FLT3+/-), and more specifically, VEN-resistant (VEN-r) AML with a CD33+ monocytic phenotype (Pei, 2020). The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) is a pivotal safeguard, selectively shielding healthy EMCN+ Hematopoietic Stem Cells and Progenitor Cells (HSCs/PCs) from potential off-tumor toxicity. Previously, SENTI-202 exhibited remarkable efficacy in targeting and eliminating CD33 and/or FLT3 expressing AML cell lines, while concurrently safeguarding healthy HSCs/PCs (Garrison et al., ASH, 2022). To further validate the efficacy and broaden the utility of SENTI-202, we conducted assessments on clinically relevant preclinical AML models, along with VEN-r AML patient-derived xenograft (PDX) models. We first assessed the in vitro cytotoxicity and cytokine profiles of CAR-NK cells possessing the SENTI-202 OR Gate and crIL15 against primary R/R AML patient samples (n=11), where we observed significantly higher cytotoxicity compared to unengineered NK-cells (68% vs 19.7%) (p=0.0005, E:T ratio 1:1 for 24h) and increased TNF-a and IFN-g secretion. To further explore CAR-NK cell anti-tumor activity, we screened 10 different PDX samples and discovered that while the median frequency of CD33 and/or FLT3 expression was 99.9% (97.48-100), individual CD33 and FLT3 expression level varied, underscoring AML proteomic heterogeneity and the clinical need for OR Gate approaches. Our initial PDX studies used clinically relevant VEN/HMA-r AML PDX cells (designated as PDX1) obtained from a patient with FLT3-ITD, GATA2, and NRAS mutations, who initially responded to VEN/decitabine treatment but later relapsed. To facilitate in vivo tracking of AML and disease progression, the VEN-r PDX cells were engineered with Akaluc, an exceptionally brighter luciferase compared to conventional luciferase. Following confirmation of AML engraftment in bone marrow through bioluminescence imaging (BLI), the CAR-NK cells, along with unengineered NK-cells, were infused at a dose of 30 million cells per infusion, once a week for three consecutive weeks. The persistence of the CAR-NK cells was detectable for up to 2 weeks after the last infusion. Notably, the infusion of CAR-NK cells resulted in the clearance of bone marrow (BM) disease as evident from BLI one week after the second infusion of CAR-NK cells while unengineered NK-cells failed to clear bone marrow disease. Assessment of CAR-NK cell efficacy in two different AML PDX models is ongoing, along with additional in vitro and in vivo studies that further demonstrate the ability of the SENTI-202 NOT EMCN Logic Gate to significantly protect healthy HSCs from off-tumor toxicity. In conclusion, CAR-NK cells expressing the SENTI-202 OR Gate and crIL15 demonstrate robust anti-tumor activity against primary AML cells within in vitro cytotoxicity assays and in vivo AML PDX models, including VEN-r AML. These promising preclinical results highlight the potential of SENTI-202 as a targeted and selective therapy for AML, offering a new avenue for overcoming the challenges posed by the heterogeneity of the AML proteomic landscape.

Targeting RARA overexpression with tamibarotene, a potent and selective RARα agonist, is a novel approach in AML.

A superenhancer at the retinoic acid receptor alpha (RARA) gene is associated with RARA mRNA overexpression in ∼30% of non-acute promyelocytic leukemia acute myeloid leukemia (AML) and in ∼50% of myelodysplastic syndromes (MDS). RARA overexpression is an actionable target for treatment with tamibarotene, an oral potent and selective RARα agonist. Sensitivity to the RARα agonist tamibarotene was demonstrated in RARA-high but not RARA-low preclinical AML models. The combination of oral tamibarotene plus azacitidine was evaluated in a phase 2 clinical study in 51 newly diagnosed unfit patients with AML identified as RARA-positive (n= 22) or RARA-negative (n= 29) for RARA mRNA overexpression in peripheral blasts using a blood-based biomarker test. In 18 response-evaluable RARA-positive patients, complete remission (CR)/CR with incomplete hematologic recovery rate was 61%, CR rate was 50%, and time to initial composite CR was rapid at 1.2 months. Transfusion independence was attained by 72% of RARA-positive patients. In contrast, 28 response-evaluable RARA-negative patients had response rates that were consistent with azacitidine monotherapy. Tamibarotene in combination with azacitidine was well tolerated. The majority of nonhematologic adverse events were low grade and hematologic adverse events were comparable to single-agent azacitidine, demonstrating that there was no additional myelosuppression when tamibarotene was combined with azacitidine. These results support further evaluation of tamibarotene-based treatment strategies in patients with AML or MDS with RARA overexpression to provide a targeted approach with the goal of improving patient outcomes. This trial was registered at www.clinicaltrials.gov as #NCT02807558.

Abstract 4950: Discovery of potent and selective CK1α molecular degrader with a favorable safety profile for acute myeloid leukemia and solid tumors

Abstract CK1α, an isoform of casein kinase 1 (CK1), promotes tumor growth by enhancing negative effects of MDM2 and MDMX on p53. Unlike other cancer types, the frequency of TP53 mutations is relatively low (< 20%) in acute myeloid leukemia (AML). Therefore, promoting apoptosis by activation of p53 pathway has been a promising strategy for treatment of AML with wild-type (WT) TP53. However, preclinical activities of p53 activators have not been fully translated into clinic, partly due to their on-target hematological toxicities. We hypothesized that inhibition of CK1α can be an alternative strategy. To develop a small molecule targeting CK1α, we identified a novel CK1α-selective molecular glue degrader (MGD), PIN-A1 through screening of compounds hijacking cereblon (CRBN) E3 ligase. Here, we report its robust pharmacological activities in various preclinical AML models as a single agent or in combination with other targeted therapies along with its superior safety profile to MDM2 inhibitors. First, we demonstrated PIN-A1 can form a ternary complex with CK1α and CRBN using TR-FRET assay. Selective degradation of CK1α by PIN-A1 was also confirmed by global proteomics analysis. PIN-A1 induced rapid degradation of CK1α in human AML MV-4-11 cells harboring WT TP53, followed by increasing the level of p53 protein and its downstream target genes, ultimately leading to apoptotic cell death. Consistent with these results, preferential sensitivity of AML cells with WT TP53 to the CK1α degrader was demonstrated in screening assay of broad panel of liquid cancer cell lines. When combined with other targeted therapies agents used for AML, PIN-A1 displayed strong synergistic effects in cell killing. To evaluate its pharmacological activity in vivo, PIN-A1 was tested in a xenograft model engrafted with MV-4-11 and a dissemination model of MOLM-14, respectively. Systemic administration of PIN-A1 alone resulted in robust antitumor activity consistent with > 90% CK1α degradation in tumor tissues, comparably achieved at a much lower dose in combo treatments. Importantly, animals remained tumor-free (5/8) in the MV-4-11 model or prolonged survival in the disseminated model even after they were no longer on the combination treatment of PIN-A1 with a targeted agent X. Lastly, while PIN-A1 had negligible effects on the viability of human PBMC with minimal activation of the p53 pathway at an efficacious dose range for MV-4-11 (Therapeutic index= 8,333), agent X induced p53 pathway and enhanced anti-leukemic activity to a similar extent in two cell types (Therapeutic index=4). Also, we found that the combination treatment of PIN-A1 with agent X had a substantial effect on several solid tumors driven by enhancing the stability and activity of p53. In summary, these results suggest that a CK1α MGD with its favorable safety profile has a potential to become an effective treatment option for advanced AML and solid tumors. Citation Format: Jinuk Kong, Jisu Park, Heekyu Lee, Hyunseok Kang, Deokjae Lee, Hyunsun Jo. Discovery of potent and selective CK1α molecular degrader with a favorable safety profile for 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 4950.

Preclinical evaluation of the ROCK1 inhibitor, GSK269962A, in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with high mortality that urgently requires new treatments. ROCK1 plays an essential role in regulating growth and survival in AML cells. In this study, we evaluated GSK269962A, a selective ROCK1 inhibitor, in preclinical models of AML. Compared with solid tumors, GSK269962A selectively inhibited cell growth and clonogenicity of AML cells. Furthermore, GSK269962A arrested AML cells in the G2 phase and induced apoptosis by regulating multiple cell cycle- and apoptosis-associated proteins. Strikingly, GSK269962A could eliminate leukemia cells from bone marrow, liver, and spleen in an animal model of AML and significantly prolong mouse survival. Mechanistically, GSK269962A could inhibit the growth of AML by blocking ROCK1/c-Raf/ERK signaling pathway. Notably, a correlation was found between the expression levels of ROCK1 protein and the sensitivity of GSK269962A in AML. These data highlight the potential role of ROCK1 as an attractive target for treating AML, as well as the potential of GSK269962A for use in clinical trials of AML.

The Multi-CDK Inhibitor Dinaciclib Shows Synergistic Activity with the BET Inhibitor PLX51107 and Reverses BET Resistance through Inhibition of Canonical Wnt Signaling in Acute Myeloid Leukemia (AML)

PLX51107 is a novel BET inhibitor (BETi) with antineoplastic activity in pre-clinical models of both aggressive B-cell malignancies and AML. Differentiating it from other BET inhibitors currently under investigation, PLX51107 has a unique binding site in the acetylated lysine binding pocket of BRD4. Through interaction with pTEFb, which is comprised of CDK9 and several cyclin T1 heterodimers, BRD4 promotes phosphorylation of RNA polymerase II, ultimately influencing gene transcription. Dinaciclib is a potent inhibitor of multiple cyclin dependent kinases including CDK9 and has been shown to have pre-clinical activity in AML. Therefore, we hypothesized that PLX51107 would have synergistic activity when combined with dinaciclib. We found synergistic effects on proliferation in primary AML cells when PLX51107 was combined with dinaciclib in stromal co-cultures across a range of dose combinations below the IC50 of either compound alone. Synergistic activity was also present in cytokine support culture conditions, colony forming assays, and synergy assays utilizing COMbenefit software. Interestingly, the highly conserved canonical β-catenin dependent Wnt signaling pathway is a known BETi resistance mechanism in AML. Therefore, destruction of β-catenin and inhibition of Wnt signaling could represent a novel therapeutic target to circumvent development of BET-mediated resistance. We found dinaciclib treatment reduced protein levels of total β-catenin in primary AML cells and AML cell lines (Figure 1A). Phosphorylated β-catenin and non-phosphorylated "active" β-catenin protein levels were also reduced following dinaciclib treatment (Figure 1A). In addition, mRNA expression of multiple components of the Wnt pathway, including the Wnt co-receptor LRP6, AXIN2, FZD1, and many Wnt target genes were significantly decreased following dinaciclib and combination treatment. Moreover, in a luciferase based Wnt reporter cell line, we found dinaciclib significantly reduced Wnt3a induced luciferase activity, suggesting a direct effect on β-catenin mediated transcription of Wnt target genes. Collectively, this data suggests dinaciclib has multiple inhibitory effects on canonical β-catenin dependent Wnt signaling. We found combination treatment of PLX51107 and dinaciclib produced synergistic reduction in expression of both Wnt signaling components and Wnt target genes. Conversely, β-catenin and Wnt target gene expression levels were either not significantly changed or even increased when treated with PLX51107 alone. In order to examine the effects of dinaciclib in the setting of BET resistance, we developed PLX51107 resistant MOLM-13 and MV4-11 AML cell lines via serial drugging. These resistant cell lines remained sensitive to dinaciclib treatment and to cytotoxic chemotherapy drugs including cytarabine and etoposide while demonstrating resistance to PLX51107 via Annexin PI and MTS assays. We found sensitivity to dinaciclib treatment in these resistant cell lines remained unaffected by BETi resistance and continued to demonstrate inhibition of Wnt signaling, similar to the effects seen in parental cells. Therefore, dinaciclib represents a novel strategy to overcome potential therapy emergent BETi resistance. We next examined the efficacy of PLX51107 in combination with dinaciclib in vivo, utilizing luciferase labeled MOLM-13 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 42 days) compared to vehicle treated control animals (median 28 days, p< 0.001). Weekly intraperitoneal 10 mg/kg dinaciclib did not significantly improve survival (median 30 days, p=0.09) in MOLM-13 xenograft mice. However, mice receiving combination therapy survived a median of 56.5 days, significantly longer than with either drug alone (p<0.05) (Figure 1B). We also found decreased disease burden by bioluminescence and by histopathology in combination treated animals, compared to both vehicle and single agent controls. In conclusion, we have demonstrated in vitro and in vivo synergistic activity of the BET inhibitor PLX51107 in combination with dinaciclib in pre-clinical models of AML and have identified novel inhibition of Wnt / β-catenin signaling by dinaciclib, thereby providing a strategy to circumvent resistance to BET inhibitors in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Novel Bispecific Innate Cell Engager (ICE®) AFM28 Efficiently Directs Allogeneic NK Cells to CD123+ Leukemic Stem- and Progenitor Cells in AML

Introduction: Acute myeloid leukemia (AML) is a common hematologic malignancy characterized by expansion of undifferentiated myeloid cells in the bone marrow (BM) and peripheral blood. Although AML can be treated with curative intent, residual leukemic stem cells (LSCs) are frequently reservoirs for relapse, which is associated with a poor prognosis. Moreover, especially elderly patients are often not eligible for intensive treatment regimen. Therefore, there is a high need for novel treatment strategies, which are capable of eliminating LSCs in order to induce long-term remissions, suppress measurable residual disease (MRD) and prevent relapse. A promising emerging approach is the adoptive transfer of allogeneic natural killer (NK) cells, which was shown to be clinically active and could induce complete remissions in relapsed/refractory (R/R) AML. Innate Cell Engagers (ICE®) may further augment the activity of NK cell therapies, in particular against LSCs and hold the potential to increase frequency and durability of responses. The tetravalent bispecific CD123/CD16A ICE®, AFM28, directs the cytotoxic activity of CD16A-expressing NK cells towards CD123-expressing (CD123+) AML blasts and LSCs. In this study, we evaluated the efficacy of AFM28 in pre-clinical models of AML. Methods: Antibody-dependent cell-mediated cytotoxicity (ADCC) data were obtained by a 4h calcein release assay using AML cell lines and primary human NK cells (E:T ratio 2.5:1) in the presence of titrated antibodies. CD64 and CD123 expression levels on AML cell lines were evaluated by flow cytometry. Intracellular FACS was used to detect STAT5/pSTAT5. Proliferation of TF-1 cells in the presence of IL-3 or GM-CSF was measured via CellTiter Glo Assay (Promega). Using primary BM samples of n=6 AML patients and n=4 healthy donors, ADCC assays and colony-forming unit (CFU) assays were performed. BM-MNC (ADCC assay) or enriched CD34+ cells (CFU assay) and allogeneic NK cells (E:T ratio 1:1) were treated with AFM28 or an Fc-enhanced anti-CD123 antibody comparator at different concentrations of AFM28 (0-500 or 0/10/100/1000 pM, respectively) for 24h. Subsequently, ADCC assays were analyzed by flow cytometry and CFU assays were evaluated after 7-14 days. Systemic anti-tumor activity of AFM28 in vivo was assessed using in-life imaging (BLI) in a T cell-depleted huFcgR-C57BL6 transgenic mouse model against intravenously administered luciferase-transfected murine AML cells expressing human CD123 as tumor antigen. Results: Pre-clinical in vitro models using a panel of AML cell lines showed efficacious ADCC against CD123+ tumor cells by allogeneic NK cells in the presence of AFM28. ADCC induction by AFM28 was independent of leukemic cell mutational profiles and even targeted cells with low levels of CD123 surface expression. Of note, expression of the high-affinity IgG receptor CD64 on AML cell lines completely abolished ADCC induction by an Fc-enhanced anti-CD123 antibody whereas AFM28 also showed high ADCC efficacy against all tested CD64+ cell lines. In addition, AFM28 exerted an NK cell-independent inhibitory effect on the IL-3-induced phosphorylation of STAT5 and abolished proliferation of CD123+ TF-1 cells, indicating an antagonistic effect on IL-3R signaling. In ex vivo models using primary AML patient cells, AFM28 demonstrated strong ADCC mediated by allogeneic NK cells. CFU assays of patient-derived AML BM samples performed subsequent to incubation with allogeneic NK cells with and without AFM28 showed significantly reduced numbers of outgrowing colonies in AFM28 treated samples, indicating that AML LSCs and progenitor cells were eliminated. In the in vivo efficacy study with huFcgR-C57BL6 mice, treatment with AFM28 at 15 mg/kg b.w. led to complete inhibition of tumor growth in 5/5 animals throughout a treatment period of 42 days. In contrast, all untreated control mice (6/6) developed systemic disease. Conclusion: AFM28 induced highly potent and selective lysis of CD123+ leukemic cells, including LSCs and progenitor cells, by allogeneic NK cells in vitro and in vivo. The capacity to eradicate LSCs holds the promise for durable responses and the potential for long-term remissions in patients with R/R AML. AFM28 is currently being prepared for a first-in-human clinical investigation as monotherapy and in combination with allogeneic NK cells.

A Potent Small Molecule Inhibitor of FLT3, PHI-101 Overcomes Resistance in Acute Myeloid Leukemia: Efficacy and PK/PD Profile in Phase 1 First in Human Study

Background: Mutations in FMS-like tyrosine kinase 3 (FLT3) are found in approximately 25-35% of acute myeloid leukemia (AML) patients and are often associated with relapse and poor prognosis. PHI-101 is an orally active, potent small molecule Type I FLT3 tyrosine kinase inhibitor (TKI) developed to overcome resistance in AML. It selectively inhibits both FLT3-ITD mutations and a wide variety of FLT3 point mutations in TKD in preclinical AML models. PHI-101 showed increased anti-leukemic activity compared to gilteritinib in both in vitro and in vivo studies with AML patient-derived primary leukemic cells. Phase 1 global clinical trials for PHI-101 (NCT04842370) against refractory and relapsed (R/R) AML are currently ongoing to assess the overall safety and efficacy. Study Design and Methods: Eligible subjects, regardless of FLT3 mutation status, were enrolled at each dose level in phase 1a to evaluate dose-limiting toxicities (DLTs) during the 28-day daily dosing period according to an accelerated 3+3 design. A dose escalation to the next dose level proceeds upon the recommendation of the safety monitoring committee. Blood samples from patients were collected to evaluate the concentration of PHI-101 in plasma during the 24-h observation period after the initial dose (cycle1, day1) and after multiple doses (cycle1, day15). A pharmacodynamic evaluation of serial plasma samples via a plasma inhibitory assay (PIA) was used to assess the FLT3-inhibitory activity of PHI-101 in FLT3-mutant cell lines. The variant allele frequency (VAF) analysis for FLT3-ITD, FLT3-TKD and other AML-related mutant in the population was performed using next-generation sequencing (NGS) technology in accordance with instructions for HEMEaccuTestTM IVD kit, with bone marrow aspirate samples collected from the patients before and after PHI-101 treatment. Results: To date, 10 enrolled patients with R/R AML have been evaluated for safety assessment at four dose levels in the dose-escalation phase 1a clinical trial of PHI-101, and they have not experienced any DLTs from daily doses of the 28-day cycle. Seventy percent of enrolled patients had more than 3 prior anti-leukemic treatment attempts, and four patients had relapsed or refractory disease after treatment with other FLT3 inhibitors including gilteritinib, quizartinib, or HM43239. The median percentage of blasts at the time of enrollment was 71%, and the median duration on study with PHI-101 was 83 days. After 1 cycle of PHI-101 treatment, bone marrow blasts were reduced by up to 98%, and the responses extended to patients who had relapsed after treatment with other FLT3 targeted therapeutics. Clinical benefits, including improvement of hematological parameters, were observed in five patients who harbored FLT3 mutations. PHI-101 exhibits a dose-proportional pharmacokinetic profile in patients. The median maximum concentration was reached 4-6h following dosing of PHI-101, and the accumulated concentration of PHI-101 in plasma on day 15 of treatment was 3-5 times higher than on day 1. Estimated T1/2 and Tmax did not appear to be dose dependent. The concomitant plasma inhibitory assay (PIA) and PK data demonstrated that exposure-related inhibition of FLT3 phosphorylation of at least 90% by plasma PHI-101 was seen within 6 hours after a single dose as long as the plasma concentration of PHI-101 reached at least 70 ng/mL. The patients with FLT3-ITD or TKD mutations showed clinical reductions of FLT3 mutation VAF of up to 77% and leukemic blast reductions of up to 87% during the same period. Notably, PHI-101 treatment significantly cleared the FLT3-N676K mutation by 96% in addition to FLT3-D835Y mutations. Conclusion: A dose-escalating phase 1a clinical data of PHI-101, which reflects up to cohort 4 of the study, indicates that PHI-101 generated potent FLT3 inhibition leading to encouraging anti-leukemic responses in R/R AML patients, including in those with prior FLT3 TKI therapeutic failure. PHI-101 showed good tolerance and favorable safety profile and reduced leukemic blasts significantly with 28-day dosing. PHI-101 sustained its activity to clear FLT3-ITD and/or FLT3-TKD mutations including D835Y or N676K identified in AML patients. Oral PHI-101 treatment results in a dose-dependent PK profile and clinically relevant efficacy of FLT3 phosphorylation inhibition. This trial indicates that PHI-101 is a next generation of FLT3 inhibitor effective for R/R AML patients.

Co-targeting of HDAC, PI3K, and Bcl-2 results in metabolic and transcriptional reprogramming and decreased mitochondrial function in acute myeloid leukemia

Despite the recently approved new therapies, the clinical outcomes of acute myeloid leukemia (AML) patients remain disappointing, highlighting the need for novel therapies. Our lab has previously demonstrated the promising outlook for CUDC-907, a dual inhibitor of PI3K and HDAC, in combination with venetoclax (VEN), against AML both in vitro and in vivo at least partially through suppression of c-Myc. In this study, we further elucidated the mechanism of action of the combination in preclinical models of AML. We demonstrated that the combination significantly reduced primary AML cell engraftment in immunocompromised mice. RNA sequencing and metabolomics analyses revealed that the combination reduced the levels for mRNAs of key TCA cycle genes and metabolites in the TCA cycle, respectively. This was accompanied by a reduced oxygen consumption rate (OCR), demonstrating that the combination suppressed oxidative phosphorylation (OXPHOS). Metabolomics analyses revealed that a large number of metabolites upregulated in AraC-resistant AML cells could be downregulated by the combination. CUDC-907 synergized with VEN in inducing apoptosis in the AraC-resistant AML cells. In conclusion, the CUDC-907 and VEN combination induces metabolic and transcriptomic reprograming and suppression of OXPHOS in AML, which provides additional mechanisms underlying the synergy between the two agents.

59 (PB049) - PIN-A1, a novel Casein Kinase 1α-selective molecular glue degrader, demonstrates strong antitumor activity via activation of the p53 pathway in preclinical models of AML with a favorable safety profile

Casein kinase 1α (CK1α) reportedly promotes tumor growth by increasing the negative effects of MDM2 and MDMX on p53. Unlike in many other cancer types, the frequency of TP53 mutations is relatively low (<20%) in acute myeloid leukemia (AML). Therefore, activating the p53 pathway has been considered an attractive strategy to treat the disease with wild-type (WT) TP53. However, promising preclinical activities of p53 activators represented by MDM2 inhibitors have not been fully translated into clinical efficacy, partly due to their on-target hematological toxicities. To develop a drug targeting CK1α, we screened a series of compounds hijacking cereblon (CRBN) E3 ligase and identified a novel CK1α-selective molecular glue degrader (MGD), PIN-A1. Here, we report its robust pharmacological activities in various preclinical models of AML and its superior safety profile to MDM2 inhibitors. First, PIN-A1’s activity against CK1α as a MGD was demonstrated by the formation of ternary complex of PIN-A1-CK1α-CRBN using TR-FRET assay and with the inhibitors of proteasome/neddylation, respectively. Selective degradation of CK1α by PIN-A1 was also confirmed by global proteomics analysis. As predicted, PIN-A1 induced rapid degradation of CK1α protein in human AML MV-4–11 cells with WT TP53, which was followed by increases in the levels of p53 protein and its downstream target genes, ultimately leading to apoptotic cell death. Consistent with these results, screening in a broad panel of liquid cancer cell lines demonstrated preferential sensitivity of AML cells with WT TP53 to the CK1α degrader. When combined with cytarabine or other targeted therapies in AML such as venetoclax (Bcl2i), AMG232 (MDM2i), or gilteritinib (FLT3i), PIN-A1 displayed strong synergistic effects in cell killing. To evaluate its pharmacological activity in vivo, PIN-A1 was tested in a s.c xenograft model of MV-4-11 and a dissemination model of MOLM-14, respectively. Systemic administration of PIN-A1 alone via i.p resulted in robust antitumor activity with >90% CK1α degradation in tumors, which was comparably achieved at a much lower dose in combo treatments. Importantly, animals remained tumor-free (5/8) in the MV-4-11 model or survived longer in the MOLM-14 model even after they were off the combination treatment of PIN-A1 with AMG232. Lastly, while PIN-A1 had minimal effects on the viability and the p53 pathway in human PBMC at efficacious doses for MV-4-11 (Therapeutic index [TI] = >8000) despite >90% CK1α degradation, AMG232 induced the p53 pathway and reduced viability to a similar extent in both cell types (TI = 4). Taken together, these results suggest that a CK1α MGD with its favorable safety profile has a potential to become an effective treatment option for advanced AML. Current activities are focused on more advanced MGD analogs with superior oral systemic exposure. No conflict of interest.

Potent preclinical activity of FLT3-directed chimeric antigen receptor T-cell immunotherapy against FLT3- mutant acute myeloid leukemia and KMT2A-rearranged acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged ALL, which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2A-rearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2Arearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3- mutant AML and KMT2A-rearranged ALL which is poised for further investigation and clinical translation.

C-Myc plays a critical role in the antileukemic activity of the Mcl-1-selective inhibitor AZD5991 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a low 5-year overall survival rate of 29.5%. Thus, more effective therapies are in need to prolong survival of AML patients. Mcl-1 is overexpressed in AML and is associated with poor prognosis, representing a promising therapeutic target. The oncoprotein c-Myc is also overexpressed in AML and is a significant prognostic factor. In addition, Mcl-1 is required for c-Myc induced AML, indicating that c-Myc-driven AML harbors a Mcl-1 dependency and co-targeting of Mcl-1 and c-Myc represents a promising strategy to eradicate AML. In this study, we investigated the role of c-Myc in the antileukemic activity of Mcl-1 selective inhibitor AZD5991 and the antileukemic activity of co-targeting of Mcl-1 and c-Myc in preclinical models of AML. We found that c-Myc protein levels negatively correlated with AZD5991 EC50s in AML cell lines and primary patient samples. AZD5991 combined with inhibition of c-Myc synergistically induced apoptosis in AML cell lines and primary patient samples, and cooperatively targeted leukemia progenitor cells. AML cells with acquired resistance to AZD5991 were resensitized to AZD5991 when c-Myc was inhibited. The combination also showed promising and synergistic antileukemic activity in vitro against AML cell lines with acquired resistance to the main chemotherapeutic drug AraC and primary AML cells derived from a patient at relapse post chemotherapy. The oncoprotein c-Myc represents a potential biomarker of AZD5991 sensitivity and inhibition of c-Myc synergistically enhances the antileukemic activity of AZD5991 against AML.