Acute Myeloid Leukemia Xenograft Models Research Articles

Discovery of a Novel Orally Bioavailable FLT3-PROTAC Degrader for Efficient Treatment of Acute Myeloid Leukemia and Overcoming Resistance of FLT3 Inhibitors.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis-targeting chimeras (PROTACs) represent a promising approach to eliminate the resistance of FLT3 inhibitors. However, due to the poor druggability of PROTACs, the development of orally bioavailable FLT3-PROTACs faces great challenges. Herein, a novel orally bioavailable FLT3-ITD degrader A20 with excellent pharmacokinetic properties was discovered through reasonable design. A20 selectively inhibited the proliferation of FLT3-ITD mutant acute myeloid leukemia (AML) cells and potently induced FLT3-ITD degradation through the ubiquitin-proteasome system. Notably, oral administration of A20 resulted in complete tumor regression on subcutaneous AML xenograft models. Furthermore, on systemic AML xenograft models, A20 could completely eliminate the CD45+CD33+ human leukemic cells in murine and significantly prolonged the survival time of mice. Most importantly, A20 exerted significantly improved antiproliferative activity against drug-resistant AML cells compared to existing FLT3 inhibitors. These findings suggested that A20 could serve as a promising drug candidate for relapsed or refractory AML.

Abstract CT089: A phase 1 multi-center, open label, dose escalation study to evaluate the safety, pharmacokinetics (PK) and anti-tumor activity of T-1301, a novel small molecular multi-target kinase inhibitor, in patients with advanced solid tumors

Abstract Background: T-1301 is a novel small molecule multi-target kinase inhibitor, it also exhibited potent inhibitory activity against both wild type and mutant forms of various kinases, i.e., ABL1, FLT3, c-KIT, PDFGRA, RET, and TRKA. In in vitro studies, T-1301 effectively inhibited growth of acute myeloid leukemia (AML) as well as gastrointestinal stromal tumor (GIST) cells carrying mutations that are resistant to KIT-targeting tyrosine kinase inhibitors (TKIs) in clinical use. In animal studies, tumor regressions were observed in AML and GIST xenograft models, while growth inhibition were observed in various other types of solid tumor models. Method: This is an open label, multi-centered Phase 1 dose escalating study (NCT05156203). The primary objectives are to establish the maximal tolerated dose (MTD) and recommended Phase 2 dose (RP2D), and to assess the safety and tolerability of T-1301. The secondary objectives are to evaluate the pharmacokinetics (PK) and therapeutic response after receiving treatment. Eligible Patient should have histologically and cytologically confirmed advanced solid tumors (including lymphoma) with measurable or evaluable target lesion(s) per RICEST v1.1 and refractory to or without standard treatments, ECOG performance status 0-1, and adequate organ function. The dose escalation begins with accelerated titration and switch to the Bayesian Optimal Interval (BOIN) design when either one of the following events is observed in Cycle 1: the first instance of DLT, or any Grade 2 or higher drug-related adverse event (AE) in any patient. Then, at least 2 additional subjects are treated at the current dose level and the dose escalation will follow the Bayesian Optimal Interval (BOIN) design with cohorts of size 3-6.T-1301 is orally administered daily in a 28-day cycle (a 21-day consecutive dosing followed by a 7-day rest). The dosing schedule is once daily (QD) at the initial dose level and could be QD or twice daily starting with the second dose level according to the safety review committee (SRC) decision after reviewing the safety and PK data from each dose level. AEs are assessed according to CTCAE v5.0 and occurrence of dose limiting toxicity (DLT) is determined during the first cycle of treatment. The safety findings are reviewed by the SRC, which will determine the MTD and RP2D. Tumor response evaluation is performed after 2 cycles of treatment and assessed according to RECIST v1.1 criteria. For those who benefit from the study medication after 2 treatment cycles, intra-subject dose escalation for multiple times is allowed in extension study period. Patient recruitment began in December 2021 and is ongoing in Taiwan. Citation Format: Jen-Shi Chen, Ming-Huang Chen, Yi-Chang Liu, Wen-Chi Chou, Nai-Jung Chiang, Hui-Ching Wang, Ta-Sen Yeh, Shih-Feng Cho, Cheng-Hsu Wang, I-Fang Lee, Li-Tzong Chen. A phase 1 multi-center, open label, dose escalation study to evaluate the safety, pharmacokinetics (PK) and anti-tumor activity of T-1301, a novel small molecular multi-target kinase inhibitor, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT089.

Abstract 6323: Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models

Abstract Background: CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012 anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential. Results: CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012 CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012 co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised murine models. Conclusion: CB-012, the first allogeneic anti-CLL-1 CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044). Citation Format: Brian Francica, Elizabeth Garner, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Erin Kelly, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Peter Lauer, Justin Skoble, Steven Kanner. Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6323.

Abstract 6728: Preclinical development of QL325, a novel T cell engager targeting CLEC12A-positive AML

Abstract Despite advancement in targeted therapies, treatment options for patients with acute myeloid leukemia (AML) that provide a tolerable and durable response remain elusive. We have developed a CD3 bispecific antibody, QL325, targeting CLEC12A, a c-type lectin protein that is highly expressed on AML blasts. Normally, CLEC12A expression is mostly confined to the CD34+CD38- progenitor population and largely absent on normal and regenerating bone marrow stem cells. Our bispecific antibody is configured in a 2 + 1 format, with bivalent avidity binding to CLEC12A, monovalent low affinity binding to CD3, and an effector-null Fc for extending half-life. In our preclinical studies, QL325 induced CLEC12A -dependent activation and proliferation of T cells. Redirected T cell-dependent cytotoxicity mediated by QL325 was restricted to AML cancer cells expressing CLEC12A. QL325 demonstrated dose dependent suppression of tumor growth in xenograft models of AML, significantly more potent than a clinical benchmark antibody. In non-human primates, QL325 was tolerated up to 0.03 mg/kg for IV dosing and under 0.1 mg/kg for SC dosing, well above the predicted effective therapeutic dose. A phase 1 clinical trial is currently ongoing to determine the safety and early efficacy in patients with refractory or relapsed AML. Citation Format: Shenda Gu, Hieu V. Tran, Shihao Chen, Ke Liu, Xin Zhang, Linchao Jia, Ruixue Ma, Jiayu Fu. Preclinical development of QL325, a novel T cell engager targeting CLEC12A-positive AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6728.

Abstract 2363: Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99-A192 ScFv-ELP nanoparticle treatment

Abstract CD99 is a transmembrane protein overexpressed in acute myeloid leukemia (AML), presenting a potential novel therapeutic target. Our previously developed anti-CD99-A192, comprising scFv and elastin-like polypeptides (ELPs), showed promising anti-leukemic activity by inducing apoptosis in AML cell lines and prolonging survival in AML xenograft models. Considering CD99's expression and role in T cell activation, we proposed that anti-CD99-A192 could have a dual function: targeting leukemic cells while activating T cells. Blood samples were obtained from 20 healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with PHA and IL-2 cytokines to expand T cells for 72-96 hours. Post-expansion, T cells were treated with either anti-CD99-A192 or control anti-A192. Proliferation was evaluated via cell count and cell trace assays. The percentages of CD4+, CD8+, and CD25+ T cells, and the levels of T cell activation markers CD69 and CD38, were quantified by flow cytometry. Cytokine release profiles were assessed by the Proteome Profiler Human Cytokine Array Kit. The cytotoxic impact of the anti-CD99-A192-activated T cells on MV4-11Luc+ AML cells was measured in co-culture apoptosis and luciferase assays. Statistical comparisons utilized paired and unpaired T-tests with Holm-Sidak correction for multiple comparisons, and P value <0.05 was considered significant. T cells treated with anti-CD99-A192 displayed significant increased proliferation compared with untreated and anti-A192-treated groups at Day 5 (163.3% vs. 97% and 92.4%, p=0.042 and p=0.03, respectively). The percentage of CD4+, CD8+, and CD25+ cells remained unchanged across treated and control groups. Activation marker analysis revealed increase in % CD38+ cells in the anti-CD99-A192 group compared with the control (58.15% vs. 49.15%, p=0.0028). Among the cytokine panel, significant increases in IL-1β/IL-1F2 (11-fold change, p<0.001, adjusted p=0.006) and TNF-α (12-fold change, p=0.002, adjusted p=0.03) were observed, along with a marked decrease in IL-8 (70% reduction, p<0.001, adjusted p=0.001). GM-CSF (20-fold change, p=0.005, adjusted p=0.071) and IL-16 (2-fold change, p=0.003, adjusted p=0.051) levels also increased, though not significantly after adjustment. Co-culture assays showed greater apoptosis in MV4-11 cells at 18 hours (33.6% vs. 41.13%, p=0.015) and a reduction in viable leukemic cells at 18 hours (10.89% vs. 8.05%, p=0.034) and 48 hours (9.69% vs. 6.54%, p=0.065) in the anti-CD99-A192 compared with the control group, results were also validated by the luciferase assay (~32% reduction, p<0.001). Anti-CD99-A192 scFv-ELP nanoparticle stimulates T cell proliferation, elevates activation markers, and increases the release of pro-inflammatory cytokines. Anti-CD99-A192 culminates in heightened cytotoxicity against leukemic cells. Citation Format: Shephali Kadam, Atham Ali, Mateusz Pospiech, Sandra Onyemaechi, Yiting Meng, Kanaka Dhuri, Andrew Mackay, Houda Alachkar. Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99-A192 ScFv-ELP nanoparticle treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2363.

Abstract 3297: Discovery and evaluation of GT19870, a GSPT1/Myc molecular glue drug (MGD) with oral bioavailability for targeting c-Myc and n-Myc-addictive blood cancer and solid tumors

Abstract c-Myc and n-Myc are oncogenic transcriptional factors driving tumor initiation, progression and poor prognosis in a variety of blood cancers and solid tumors, such as acute myeloid leukemia (AML), B-cell malignancies, castration resistance prostate cancer (mCRPC), small cell lung cancer and (SCLC) and neuroblastoma. Both c-Myc and n-Myc dysregulations have been directly linked to the poor clinical outcome in some of these cancers which are addictive to deregulated Myc proteins. Therefore, it is highly warranted to discover and develop novel therapeutical agents for targeting Myc-addictive cancers. GT19870 was discovered through a SAR effort for Myc degradation with improved bioavailability (F % 92.52) from GT19630, a GSPT1/Myc degrader series 1-4by using phenotypic screenings with c-Myc-addictive HL-60 AML cells and growth factor-dependent hematopoiesis progenitor TF-1 cells, followed by c-Myc ELISA and Western blot assays. GT19870 selectively inhibited the proliferation of HL60 cells with an IC50 of 2.6 nM as compared to an IC50 of 70.03 nM (26.5-fold selectivity) and effectively degraded c-Myc protein with an IC50 of 2.54 nM and IC90 <10 nM in HL-60 cells, as compared to an IC90>30 nM in CD3/CD28-stimulated PBMC assays. GT19870, as a MGD, was confirmed to selectively degrade GSPT1, a neo-substrate, by proteomic and Western Blot assays. Further, GT19870 was tested in SCLC, triple negative breast cancer (TNBC) and brain cancer cell panels, in which GT1987 was revealed potent anti-proliferation activities with an IC50<50 nM in 9 of 19 SCLC, 8 of 13 TNBC, and 7 of 24 brain cancer cell lines tested. Importantly GT19870 was also shown potent antitumor activities with IC50 of ≦5.4 nM in 8 of 8 SCLC patient derived organoids. Furthermore, GT19870 (salt form-GT19884) was demonstrated to induce target-engaged (c-Myc and n-Myc) tumor regression in AML, SCLC, mCRPC and neuroblastoma xenograft and PDX models with in a dose ranges of 3-12 mpk/qd/po. In brief, GT19870 is an oral MGD degrading GSPT1/c-Myc/n-Myc. This novel MGD has been demonstrated target-engaged antitumor activities in vitro and in vivo with selectivity of c-Myc degradation in c-Myc-addictive cancer cells as compared to growth factor-stimulated TF-1 cells and CD3/CD28 activated PBMC. GT19870 has been progressing IND enabling stage.

Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS and AML.

Splicing modulation is a promising treatment strategy pursued to date only in splicing factor-mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we showed that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing factor 3B subunit 1 (SF3B1) H3B-8800 and E-7107. We identified drug-induced alterations in splicing, and corresponding reduced gene expression, of a number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We found that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators due to their long length and large number of exons per transcript. Furthermore, we demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators not only resulted in impaired DNA damage response and accumulation of DNA damage, but it sensitized cells to subsequent killing by poly(ADP-ribose) polymerase (PARP) inhibitors and chemotherapy and led to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML.

Abstract LB_A05: Coadministration of AZD 4573 and gilteritinib exhibits a potent activity in FLT3 inhibitor-sensitive and -resistant AML cells

Abstract Acute myeloid leukemia (AML) is caused by the sequential acquisition of mutations that regulate multiple cellular pathways, indicating that multiple pathways must be coordinately inhibited for a successful therapy in this disease. Transcriptomics analysis suggest that exposure of AML cells to the selective CDK9 inhibitor AZD4573 led to activation of multiple survival pathways including MAPK, PI3K, and STAT suggesting that disruption of these signaling pathways may potentiate this agent activity. Notably, combined treatment with AZD4573 and the FLT3 inhibitor gilteritinib resulted in a robust suppression of cell growth and a marked induction of apoptosis in various AML cell lines, particularly those with FLT3 mutations. These were associated with a marked induction of apoptosis reflected by increased Annexin V/PI positivity and cleavage in caspase 3 and its substrate PARP. Importantly, combined treatment was also highly effective in FLT3 mutants AML cells that exhibit significant resistance to various FLT3 inhibitors including gilteritinib. Combined treatment led to a profound disruption of critical survival signaling pathways including MEK/ERK, STAT, and PI3K/AKT pathways. Combined treatment also significantly disrupted the balance between the proapoptotic and anti-apoptotic Bcl-2 members as reflected by downregulation of MCL1 and upregulated of Bim protein levels. Importantly, GILT/AZD4573-induced cell death was fully prevented when Bax and Bak were knocked out using CRISPR system, suggesting that the anti-AML activity of this regimen proceeds mainly via the mitochondrial apoptotic pathway. Finally, using a systemic acute myeloid leukemia xenograft model, co-administration of gilteritinib and AZD4573 sharply reduced leukemia growth and markedly prolonged mice survival. Collectively, these findings demonstrate that dual inhibition of CDK9 and FLT3 exhibits a potent anti-leukemic efficacy in vitro and in vivo. These findings also indicate that this strategy warrants further attention in FLT3-dependent acute myeloid leukemias. Citation Format: Mohamed Rahmani, Baraa Khalid Salah, Burcu Yener, Bassam Mahboub, Steven Grant. Coadministration of AZD 4573 and gilteritinib exhibits a potent activity in FLT3 inhibitor-sensitive and -resistant AML cells [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 LB_A05.

CLL-1-Targeted Allogeneic CAR-T Cells Exhibit High on-Target Specificity and Potent Cytotoxicity in Preclinical Models of Acute Myeloid Leukemia

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Human iPSC-Derived NK Cells with Knock-in of the BCL2 G101V Mutation Are Resistant to Venetoclax and Demonstrate Improved Anti-AML Activity In Vivo

Treatment options for acute myeloid leukemia (AML) patients who are unfit for intensive chemotherapy are limited. Both targeted therapy and cellular therapy have been investigated, typically as separate treatments. For patients with AML unfit for intensive induction chemotherapy, venetoclax, a BCL-2 inhibitor, containing regimens have significantly improved treatment outcomes. However, patients treated long-term with venetoclax can develop resistance. Based on the characterization of the BCL2 G101V mutation that mediates resistance to venetoclax, we investigated the ability of homozygous BCL2 G101V knock-in in induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells to promote resistance to venetoclax in these effector cells to enable concurrent treatment with venetoclax and the engineered iPSC-derived NK cells that we hypothesize will provide improved NK cell-mediated killing of AML. Our group has pioneered the production of genetically modified NK cells from human iPSCs. iPSCs provide an advantage of a stable platform for genome engineering and previous studies demonstrate we can engineer iPSCs to express or delete genes of interest to derive genetically modified iPSC-NK cells with improved anti-tumor activity. To generate iPSC-derived NK cells with resistance to venetoclax, we employed CRISPR-Cas9 technology to knock-in the BCL2 G101V mutation (BCL2 G101V) in iPSCs. iPSCs homozygous for BCL2 G101V were selected and differentiated to NK cells. Homozygous BCL2 G101V engineered iPSC-NK cells demonstrated resistance to venetoclax compared to wildtype (WT) iPSC-NK cells in vitro. BCL2 G101V iPSC-NK cells were 94-fold more resistant to venetoclax compared to wildtype iPSC NK cells with an EC50 of 6018 nM, above the serum levels of patients receiving venetoclax. Analysis of cell surface proteins demonstrated that both sets of iPSC-NK cells had a typical NK cell phenotype with no differences in expression of receptors analyzed. There was no difference seen in cytotoxicity against K562 tumor cells between BCL2 G101V iPSC-NK cells and WT-iPSC NK cells. Additional functional analyses demonstrated that activity of the BCL2 G101V iPSC NK cells was preserved upon exposure to venetoclax. Cytotoxic activity, as measured by CD107a expression on WT iPSC-NK cells stimulated by MOLM13 AML tumors cells, was reduced nearly 3-fold upon addition of venetoclax. In contrast, activation of BCL2 G101V iPSC NK cells was not significantly affected by addition of venetoclax. This resistance to venetoclax was confirmed in longer term 36-hour Incucyte cytotoxicity assays where BCL2 G101V iPSC-NK cells demonstrated more than 10-fold increase in anti-AML activity compared to the WT-iPSC-NK cells. We then conducted in vivo studies to test the WT and engineered NK cells with and without addition of venetoclax in an AML xenograft model using NSG mice (Figure Panels A-D). These studies used MOLM13 AML cells made resistant to venetoclax and demonstrated that without venetoclax treatment, both the WT and the BCL2 G101V iPSC NK cells mediate effective anti-AML killing. With addition of venetoclax, the WT NK cells were unable to effectively kill the AML cells while the BCL2 G101V iPSC cells mediated potent anti-AML activity and demonstrated significantly improved survival when the NK cells are given in combination with venetoclax. Specifically, in this system, the mice treated with WT iPSC-NK cells had a median survival of 22.8 days while the median survival of mice treated with the BCL2 G101V iPSC NK cells was not reached (p<0.01) Intriguingly, BCL2 G101V iPSC NK cells also demonstrated improved activity against MOLM13 cells resistant to venetoclax. Together our results demonstrate that iPSC-NK cells can be engineered to generate venetoclax-resistance for use in combination with concurrent venetoclax therapy to markedly improve treatment of AML. Furthermore, this work demonstrates that novel drug resistance mechanisms can be introduced via genome engineering into iPSC-derived NK cells as a new strategy to produce improved cell products for “off-the-shelf” therapy.

Komet-008: A Phase 1 Study to Determine the Safety and Tolerability of Ziftomenib Combinations for the Treatment of KMT2A-Rearranged or NPM1-Mutant Relapsed/Refractory Acute Myeloid Leukemia

Background and Significance: Relapsed or refractory (R/R) acute myeloid leukemia (AML) with nucleophosmin 1-mutations ( NPM1-m) or KMT2A-rearrangment ( KMT2A-r) remain a high unmet need. Ziftomenib is a potent and selective inhibitor of the interaction between menin and mixed-lineage leukemia (MLL) lysine[K]-specific methyltransferase 2A ( KMT2A), which drives leukemogenesis in these subtypes. Preclinical data have demonstrated ziftomenib's ability to target multiple types of menin-dependent AML clones, and that the antitumor activity of ziftomenib and other menin inhibitors in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models of KMT2A-r and NPM1-m AML is enhanced by the addition of FLT3 inhibitors such as gilteritinib and quizartinib. An ongoing Phase 1/2 study (KOMET-001) in R/R AML has demonstrated that ziftomenib monotherapy is safe and well tolerated and that the clinical activity at the recommended phase 2 dose (RP2D) is robust, as the rates of response were not affected by the presence of downstream co-mutations, such as FLT3 and IDH1 or IDH2. Given the monotherapy activity seen to date, the development of ziftomenib in combination with standard-of-care (SOC) chemotherapies or FLT3 inhibitors may provide increased clinical benefit for R/R AML patients with KMT2A-r or with NPM1-m with or without concurrent FLT3-mutations ( FLT3-m) and warrants further investigation. Study Design and Methods: KOMET-008 (NCT# 05735184) is an open-label, dose escalation and expansion study to determine the safety, tolerability, and preliminary efficacy of ziftomenib when combined with SOC regimens for the treatment of either NPM1-m (Arm A) or KMT2A-r (Arm B) R/R AML. Each genetically defined arm will evaluate ziftomenib in combination with various SOC treatments in separate and independent cohorts, as outlined below: Arm A:NPM1-m R/R AML Cohort A-1: Ziftomenib plus FLAG-IDACohort A-2: Ziftomenib plus LDACCohort A-3: Ziftomenib plus gilteritinib (for NPM1-m + FLT3-m R/R AML) Arm B: KMT2A-r R/R AML Cohort B-1: Ziftomenib plus FLAG-IDACohort B-2: Ziftomenib plus LDAC The Part 1a dose escalation uses a i3+3 design, followed by dose expansion/validation in Part 1b. Key eligibility criteria include adults (≥18 years) with documented NPM1-m (±co-occurring FLT3-m) or KMT2A-r R/R AML, ECOG ≤2, prior hematopoietic stem cell transplant (HSCT) allowed, if recovered and prior gilteritinib allowed. Patients who receive an HSCT during study participation will be allowed to receive ziftomenib maintenance therapy upon recovery. The primary objectives are to establish the safety and tolerability of ziftomenib when combined with SOC therapies (ie, dose limiting toxicities, AEs per CTCAE v5.0) and to identify the RP2D of ziftomenib for each SOC combination in each genetically defined cohort. Secondary endpoints include preliminary efficacy (including MRD-negative responses and number of patients able to receive stem cell transplant) and pharmacokinetics. Exploratory end points include biomarkers for efficacy and resistance and pharmacodynamic biomarkers potentially related to the activity of ziftomenib when combined with SOC treatments in adults with R/R AML with NPM1-m or KMT2A-r.

The Mevalonate Pathway Is a Therapeutic Target in TP53 Mutant Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) with mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a median overall survival of only six months due to chemotherapy resistance. To understand biologic differences in TP53 mutant (MT) AML, as compared to wildtype (WT) AML, we performed transcriptomic analysis on sorted patient samples. Gene set enrichment analysis demonstrated significant upregulation of the cholesterol biosynthesis or mevalonate pathway in TP53 MT AML. In TP53 MT solid tumor models, the mevalonate pathway plays a key role in tumorigenesis and metastasis. We hypothesized that the mevalonate pathway is essential for chemotherapy resistance of TP53 MT AML and represents a novel therapeutic target. Methods: We developed chemoresistant, isogenic TP53 MT AML cell lines using CRISPR/Cas9 technology in the MOLM14 AML cell line. We used primary TP53 MT AML patient samples to perform colony forming unit (CFU) assays, in vitro assays with a serum substitute and cytokines, and a patient derived xenograft (PDX) model of TP53 MT AML. Results: We first sought to determine if TP53 MT AML exhibits upregulation of mevalonate pathway activity in response to cytarabine (AraC), the backbone of AML therapy. We treated our isogenic TP53 MT AML cell lines for 24 hours (h) with AraC and assessed mevalonate pathway gene expression and metabolites using qRT-PCR and liquid chromatography high resolution mass spectrometry (LC-HRMS), respectively. Only TP53 MT cell lines respond to AraC with a significant upregulation of key mevalonate pathway genes and metabolites. We then determined if TP53 MT AML is sensitized to AraC by inhibition of the mevalonate pathway with a statin. We pretreated our isogenic cell lines with 24h rosuvastatin before 24h AraC and assessed cell viability with flow cytometry with AnnexinV/7AAD staining and XTT assays. We also performed CFU assays in TP53 MT AML patient samples treated with the two drugs alone or in combination and assessed CFUs after 14 days. These studies revealed a synergistic reduction in cell viability and CFUs in TP53 MT AML by rosuvastatin in combination with AraC. We next addressed the mechanism by which a statin sensitizes TP53 MT AML to AraC. Our group and others have demonstrated enhanced mitochondrial activity is associated with AML chemoresistance. We hypothesized that mevalonate pathway byproducts, known to be crucial for mitochondrial functions, contribute to the mitochondrial response to AraC. We treated TP53 MT AML cell lines as above with rosuvastatin and AraC and used seahorse technology to measure oxidative phosphorylation (OXPHOS). We also used electron microscopy, flow cytometry of mitochondrial protein, TOM20, and qRT-PCR for mitochondrial DNA content to quantify and characterize mitochondria. These data demonstrate that TP53 MT AML cell lines, compared to WT, exhibit a significant increase in OXPHOS after 24h that is due to an increase in total mitochondrial. These effects are abrogated by pretreatment with a statin. We also validated these findings in primary TP53 MT AML patient samples in vitro. Importantly, co-treatment of the isogenic TP53 MT AML cell lines with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. Overall, this data supports a model where TP53 MT AML cells dynamically upregulate the mevalonate pathway to regulate OXPHOS and avoid DNA damage-induced cell death (Figure 1). Finally, we studied a PDX model of TP53 MT AML. Engrafted mice were treated with AraC for five days alone or in combination with high dose rosuvastatin. Mice were harvested on day eight with assessment of leukemic burden by flow, OXPHOS by seahorse and mevalonate byproducts by LC-HRMS. TP53 MT AML leukemic burden was only significantly reduced by the combination therapy. Consistent with in vitro findings, mice sacrificed three days after completing AraC continue to demonstrate enhanced OXPHOS and increased mevalonate byproducts. This response is blunted by rosuvastatin. Conclusions: These results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of this pathway in combination with chemotherapy may improve clinical responses. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells in order to elucidate novel therapeutic approaches.

Alrizomadlin (APG-115) Alone or Combined with Azacitidine (AZA) in Patients (pts) with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML) or Relapsed or Progressive Higher-Risk Myelodysplastic Syndrome (HR-MDS): Phase 1b Trial Results

*Drs. Yifan Zhai and Jianxiang Wang are co-corresponding authors. Background Pts with R/R AML and HR-MDS failing hypomethylating agents have limited therapeutic options and relatively dismal outcomes with available therapies. Investigational alrizomadlin is a novel, orally active, potent, small-molecule selective inhibitor that destabilizes the p53-MDM2 complex and activates p53-mediated apoptosis in tumor cells and has shown apoptotic activity in AML and MDS xenograft models, alone and combined with AZA. Here, we present safety and efficacy results of this therapy in these pts. Methods This open-label dose escalation and expansion trial included adults with R/R AML or relapsed/progressed HR-MDS (IPSS-R ≥ 4.5). MTD and RP2D of alrizomadlin ± AZA were determined by dose escalation. Alrizomadlin (100/150/200/250 mg) was administered orally on Days (D) 1-7 in 28-day cycles. In the combination dose escalation group, alrizomadlin (100/150/200 mg) + AZA (75 mg/m 2) was administered orally once daily on D 1-7 in 28-day cycles and at the combination RP2D during dose expansion. Responses were assessed per revised International Working Group (IWG) Response Criteria 2003, ELN 2017 classifications for AML, and IWG 2006 for MDS. Extensive pharmacokinetic (PK) analyses were performed. Results As of June 1, 2023, 29 pts were enrolled in China. In the monotherapy group, 21 pts (median [range] age, 65 [32-76] years; 76.2%, R/R AML) had a median (range) of 2 (1-10) prior lines of therapy. In the combination group, 8 pts (median [range] age of 69.5 [20-76] years; 50%, R/R AML) had a median (range) of 2 (1-4) prior lines of therapy. In both groups, common any-grade treatment-related adverse events (TRAEs; ≥ 20%) were hematologic- and gastrointestinal-related toxicities, hypokalemia, and dizziness, and most grade ≥ 3 TRAEs were hematologic toxicities. No grade ≥ 3 gastrointestinal toxicities were reported (Figure 1). One DLT, a pulmonary embolism (PE), was reported among 6 evaluable patients at the alrizomadlin 100 mg + AZA dose level: a 59-year-old pt with MDS had a prior COVID-19 infection and abnormal coagulation function before (and during) study treatment. The study investigator attributed the PE to the pt's COVID-19 infection, age, primary disease, and long-term bed rest. One pt in each arm discontinued treatment because of AEs. In pts with AML, the overall response rate (ORR) was defined as complete remission (CR) + CR with incomplete hematologic recovery (CRi) + partial response (PR) + morphologic leukemia-free state (MLFS). In pts with MDS, ORR was defined as CR + PR + marrow CR (mCR). In the monotherapy group, the ORR and CR/CRi rates among pts with R/R AML were 25% (4/16) and 18.75% (3/16), respectively. Three of 6 pts with MDS that progressed to R/R AML experienced responses, including 2 CRis and 1 MLFS. One of 10 pts with R/R AML treated with venetoclax + HMA experienced a CRi. In the monotherapy cohort, 4 pts with MDS refractory to HMAs were evaluated, of whom 2 experienced an mCR. The monotherapy RP2D was determined as 200 mg. In the combination group, 6 pts had an efficacy evaluation at least once. Among these pts, 3 of 3 with MDS experienced an mCR and 1 of 3 with R/R AML, MLFS. Among 4 pts with prior venetoclax + HMA treatment, 2 with MDS experienced an mCR and 2 with R/R AML showed a half marrow blast reduction at the end of C1 (Table 1). No PK drug-drug interaction between alrizomadlin and AZA was observed. Conclusions Alrizomadlin alone or combined with AZA demonstrated a manageable safety profile and preliminary efficacy in pts with R/R AML and HMA-refractory MDS. An antileukemic effect was observed in both monotherapy and combination settings, including a few pts whose disease failed on prior venetoclax treatment. Evaluation of the alrizomadlin + AZA cohort is ongoing. Internal study (CT.gov) identifiers: APG-115-AC101 (NCT04275518).

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

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.

Cooperative CAR targeting to selectively eliminate AML and minimize escape

Acute myeloid leukemia (AML) poses a singular challenge for chimeric antigen receptor (CAR) therapy owing to its phenotypic heterogeneity and similarity to normal hematopoietic stem/progenitor cells (HSPCs). Here we expound a CAR strategy intended to efficiently target AML while minimizing HSPC toxicity. Quantification of target expression in relapsed/refractory patient samples and normal HSPCs reveals a therapeutic window for gated co-targeting of ADGRE2 and CLEC12A: We combine an attenuated ADGRE2-CAR with a CLEC12A-chimeric costimulatory receptor (ADCLEC.syn1) to preferentially engage ADGRE2posCLEC12Apos leukemic stem cells over ADGRE2lowCLEC12Aneg normal HSPCs. ADCLEC.syn1 prevents antigen escape in AML xenograft models, outperforms the ADGRE2-CAR alone and eradicates AML despite proximate myelopoiesis in humanized mice. Off-target HSPC toxicity is similar to that of a CD19-CAR and can be mitigated by reducing CAR Tcell-derived interferon-γ. Overall, we demonstrate the ability of target density-adapted cooperative CAR targeting to selectively eliminate AML and potentially obviate the need for hematopoietic rescue.

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.

Epichaperome inhibition targets TP53-mutant AML and AML stem/progenitor cells

AbstractTP53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53–wild-type (WT) and -mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the therapeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34+ cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and -mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and -mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.

Abstract 2648: In vivo efficacy and preclinical safety profile of NN3201, an anti-cKIT antibody-drug conjugate, for treating SCLC and AML

Abstract Overexpression and activating mutations of cKIT are associated with tumor aggressiveness and poor prognosis in various cancer patients such as small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), and mastocytoma. Since small molecule inhibitors including imatinib frequently have a limited therapeutic application due to eventual drug resistance and severe adverse events, we developed the cKIT-directed antibody-drug conjugate (ADC), NN3201, for the treatment of wild-type and mutant cKIT-positive cancers. NN3201 is an ADC conjugate of the antimitotic payload MMAE to the fully human 2G4 antibody, directed against cKIT, via the cysteine re-bridging technology ThioBridge® at a drug-to-antibody ratio (DAR) of 4. NN3201 displayed preferable biophysical properties and broadly potent in vitro antitumor activities across various cKIT-positive tumor cell lines. NN3201 exhibited potent in vivo antitumor activities in cKIT-positive tumor cell line xenograft and patient-derived tumor xenograft models of SCLC and AML, representing robust and durable antitumor responses. Furthermore, in a single-dose toxicity study with cynomolgus monkey, no significant or severe clinical toxicity signs were observed in animals dosed at 1, 2, and 3 mg/kg. Even though a decrease in reticulocyte, red cell mass (hemoglobin and red blood cell), neutrophil, and monocyte counts, along with bone marrow recovery and reticulocyte compensatory mechanism, red blood cell and white blood cell recovery were observed from day 14 in the low and medium dose groups and day 21 in the high dose group, dose-dependently. These preclinical results suggest that NN3201 is a potential therapeutic agent for the treatment of cKIT-positive cancers and support the further clinical evaluation in SCLC and AML. Citation Format: Han-Jik Ko, Sung Tae Kim, Jin Gu Cho, Jin-Ock Kim, Jung Tae Lee, Sang Gyu Park. In vivo efficacy and preclinical safety profile of NN3201, an anti-cKIT antibody-drug conjugate, for treating SCLC and AML [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 2648.

Type I interferon signaling in malignant blasts contributes to treatment efficacy in AML patients

While type I interferon (IFN) is best known for its key role against viral infection, accumulating preclinical and clinical data indicate that robust type I IFN production in the tumor microenvironment promotes cancer immunosurveillance and contributes to the efficacy of various antineoplastic agents, notably immunogenic cell death inducers. Here, we report that malignant blasts from patients with acute myeloid leukemia (AML) release type I IFN via a Toll-like receptor 3 (TLR3)-dependent mechanism that is not driven by treatment. While in these patients the ability of type I IFN to stimulate anticancer immune responses was abolished by immunosuppressive mechanisms elicited by malignant blasts, type I IFN turned out to exert direct cytostatic, cytotoxic and chemosensitizing activity in primary AML blasts, leukemic stem cells from AML patients and AML xenograft models. Finally, a genetic signature of type I IFN signaling was found to have independent prognostic value on relapse-free survival and overall survival in a cohort of 132 AML patients. These findings delineate a clinically relevant, therapeutically actionable and prognostically informative mechanism through which type I IFN mediates beneficial effects in patients with AML.