Residual disease remains a major obstacle for achieving durable responses in patients treated with oncogene-targeted therapy. Drug-tolerant persister (DTP) cells emerging under treatment and persisting in residual tumors are considered to be the root of acquired resistance, yet their contribution to immune evasion in on-treatment tumors is poorly defined. Here, we show in the context of EGFR-mutant lung cancer that DTP cells actively contribute to the formation of an immunosuppressive tumor microenvironment during EGFR tyrosine kinase inhibitor (TKI) therapy. In syngeneic mouse models and in patients, EGFR TKI therapy leads to an accumulation of immunosuppressive macrophages, which is strictly treatment-dependent and fully reversible upon treatment cessation or progressive disease, respectively. Quiescent DTP cells directly drive the recruitment and immunosuppressive reprogramming of monocytes and macrophages through a YAP-driven secretome, and the DTP-reprogrammed monocytes suppress T cell proliferation and effector functions in vitro. Co-targeting YAP with a TEAD inhibitor ORM-47286 rewires the DTP secretome and inhibits macrophage reprogramming in vitro, and prevents immunosuppressive macrophage accumulation and improves the efficacy of EGFR TKI therapy in immunocompetent mouse models. Our findings highlight the previously unappreciated role of DTP cells in modulating the tumor microenvironment in on-treatment tumors, and position the treatment-induced YAP/TEAD activity in DTP cells as an important driver of adaptive immunosuppression during EGFR-targeted therapy. ### Competing Interest Statement PAJ has received consulting fees from AbbVie, Accutar Biotech, Allorion Therapeutics, AstraZeneca, Bayer, Biocartis, Boehringer Ingelheim, Chugai Pharmaceutical Co., Daiichi Sankyo, Duality, Eisai, Eli Lilly, Frontier Medicines, Hongyun Biotechnology, Merus, Mirati Therapeutics, Monte Rosa, Novartis, Nuvalent, Pfizer, Roche/Genentech, Scorpion Therapeutics, SFJ Pharmaceuticals, Silicon Therapeutics, Syndax, Takeda Oncology, Transcenta, and Voronoi; sponsored research support from AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Eli Lilly, Puma Biotechnology, Revolution Medicines, and Takeda Oncology; post-marketing royalties from a DFCI-owned patent on EGFR mutation licensed to Lab Corp; and has stock ownership in Gatekeeper Pharmaceuticals. The other authors declare no competing interests. Finnish Cultural Foundation, https://ror.org/027xav248 Research Council of Finland, 346656, 370023 Sigrid Jusélius Foundation, https://ror.org/00ckakm23 Jane and Aatos Erkko Foundation, https://ror.org/03vxy9y38 Instrumentarium Science Foundation, https://ror.org/02w907m39 Syöpäsäätiö, https://ror.org/0113k5861 Emil Aaltosen Säätiö, https://ror.org/005rt3g54

BACKGROUND:Hypomethylating agent (HMA) and the BCL-2 inhibitor venetoclax (VEN) combinations have evolved into frontline therapies for patients with acute myeloid leukemia (AML), yielding high response rates. However, most patients ultimately relapse, particularly those with TP53 mutations. We investigated mechanisms of action and therapeutic efficacy of NTX-301, a next-generation HMA. Methods used include flow cytometry-based cell viability assays, Western blot, reverse-phase protein arrays, RNA-sequencing, CyTOF single-cell mass cytometry, and methylation profiling in various therapy-resistant AML models. RESULTS:We demonstrate that NTX-301 exhibits superior efficacy compared to 5-azacytidine (5-AZA) in 5-AZA or VEN-resistant AML. It synergizes with VEN in VEN- or VEN/HMA-resistant and TP53-mutant AML blasts and stem/progenitor cells (combination index<1). NTX-301 inhibits DNMT1 and increases p73, caspase-8/activated caspase-8 levels in TP53-WT and TP53-mutant AML and activates p53 signaling. It extends survival (≥45%) in both, xenograft and PDX models. Methylation profiling revealed that NTX-301 is a more targeted HMA compared to 5-AZA, enabling suppression of functionally enriched genes/pathways. Pathway analysis of 954 commonly hypomethylated genes showed profoundly greater enrichment of Hippo signaling in NTX-301-treated compared to 5-AZA-treated cells, and enrichment of insulin signaling, VEGF pathway, and cell cycle selectively in NTX-301- but not in 5-AZA-treated cells. NTX-301-mediated Hippo signaling was validated at protein levels. CONCLUSION:Data suggest that NTX-301 exerts potent anti-leukemia activities superior to 5-AZA and synergizes with VEN in VEN-resistant and TP53-mutant AML, in part by suppressing DNMT1 and inducing DNA damage responses and apoptosis, by inducing p53 signaling and demethylating LATS1/2, thus activating Hippo signaling.

Direct targeting of the oncoprotein MYC has not yet been successful. We here report a novel dual protein degrader, GT19630, which binds directly to MYC and G1 to S phase transition protein 1 (GSPT1). GT19630 disrupts a novel feedforward loop of MYC and GSPT1, where MYC promotes transcription of GSPT1, and GSPT1 senses the stop codon of MYC to properly terminate its translation. The agent induces integrated stress response and abrogates oxidative phosphorylation through inhibition of the TCA cycle, resulting in apoptosis. GT19630 has superior activity compared to GSPT1- targeting molecular glues. GT19630 induces profound anti-proliferative effects and apoptosis at low nanomolar concentrations in a multitude of leukemia and lymphoma cell lines and primary samples, including those with TP53 mutations. GT19630 is highly active in vivo in models of therapy-resistant hematologic malignancies, including Burkitt's lymphoma, acute myeloid leukemia (AML) and multiple myeloma. CD34+ AML blasts overexpress MYC protein compared to normal hematopoietic stem/progenitor cells (HSPCs) and GT19630 induces greater cytotoxicity in AML cells compared to normal HSPCs. Further, GT19630 restores sensitivity to venetoclax and profoundly prolongs survival in vivo in venetoclax-resistant AML. GT19630 was well tolerated in humanized Crbn mice. In conclusion, our data support the development of the MYC/GSPT1 degrader GT19630 as a therapeutic strategy of MYC-driven hematologic malignancies.

ABSTRACT:Recessively inherited loss-of-function mutations in excision repair cross-complementing 6-like 2 (ERCC6L2) cause a bone marrow failure (BMF) syndrome characterized by moderate cytopenias, frequent somatic TP53 mutations, and a propensity to develop myeloid malignancies. The pathophysiology and molecular mechanisms underlying the BMF syndrome as well as its association with TP53-mutant clonal hematopoiesis and myeloid malignancies have remained poorly understood. Using novel preclinical in vitro and in vivo model systems, we demonstrate that Ercc6l2 maintains the competitive fitness of hematopoietic stem and progenitor cells (HSPCs) by mitigating replication stress. Sustained replication stress and DNA damage in Ercc6l2-deficient HSPCs cause p53 pathway activation followed by cell cycle arrest and apoptosis. Moreover, Ercc6l2 deficiency results in decreased expression of master hematopoietic regulators Runx1 and Gata1 in HSPCs. Altogether, loss of Ercc6l2 leads to reduced HSPC numbers, bone marrow hypocellularity, and cytopenias. Notably, somatic Trp53 mutations restore cellular fitness of Ercc6l2-deficient HSPCs by abrogating p53 pathway activation and restoring Runx1 and Gata1 expression, thereby correcting the BMF phenotype. However, p53 loss fails to normalize replication stress, allowing for the accumulation of DNA damage over time, which increases the likelihood for leukemic transformation. Our data uncover the pathogenesis of ERCC6L2 disease and provide a prototypic example of clonal compensation in BMF syndromes, in which somatic mutations in leukemia-associated genes, in this case TP53, transiently improve blood cell production at, however, the expense of increasing leukemogenic potential.

Chimeric Antigen Receptor (CAR) T-cell therapy has transformed cancer immunotherapy by genetically engineering T-cells to target tumor antigens. Acute myeloid leukemia (AML) presents unique challenges due to resistance mechanisms, especially in patients with TP53 loss mutations. The complex dynamics of CAR T-cell expansion remain poorly understood. The field lacks validated quantitative frameworks to systematically evaluate different CAR T-cell target constructs, such as CD33, CD123, and CD371, against resistant AML variants. We address this gap by combining mathematical modeling with in vitro assay data and Bayesian inference. We select, train, and validate a two-compartment deterministic mathematical model that describes the nonlinear dynamics of target AML and CAR T cells, accounting for expansion, killing, and exhaustion. Using Bayesian inference, we train and select the best-performing functional form for CAR T expansion and then validate it on unseen data. Our framework selects a CAR T-cell expansion model that accounts for handling time and T-cell self-interference, highlighting that expansion is a dynamic process in which target-cell handling time and T-cell crowding negatively affect T-cell expansion. Analysis of posterior parameter distributions reveals target-antigen-specific responses against TP53-deficient AML. For instance, CD33-targeting CARs have reduced attack rates against TP53-deficient cells, while CD123- and CD371-targeting CARs show moderately increased attack rates; however, the former exhibit higher death rates, and the latter have increased handling times, impeding efficacy. This target-dependent form of resistance challenges the assumption of uniform performance and reveals a unifying nonlinear expansion model for integrated, yet antigen-specific, preclinical predictions of efficacy.

DNA suffers continual damage leaving a cell with thousands of individual DNA lesions at any given moment 1–3 . The efficiency of DNA repair means that most known classes of lesion have a half-life of minutes to hours 3,4 , but whether some DNA damage can persist for longer durations remains unknown. Here, using high-resolution phylogenetic trees from 89 donors, we identified mutations arising from 832 DNA lesions that persisted across multiple cell cycles in normal human stem cells from blood, liver and bronchial epithelium 5–12 . Persistent DNA lesions occurred at increased rates, with distinctive mutational signatures, in donors exposed to tobacco or chemotherapy, suggesting that they can arise from exogenous mutagens. In haematopoietic stem cells, persistent DNA lesions, likely from endogenous sources, generated a characteristic mutational signature, so-called SBS19 13 ; occurred steadily throughout life, including in utero ; and endured for 1.5 years on average, with 15% lasting 3+ years. We estimate that a haematopoietic stem cell has, on average, ~4-5 such lesions at any moment in time, half of which will generate a mutation with each cell cycle. Overall, 16% of mutations in blood cells are attributable to SBS19, and similar proportions of driver mutations in blood cancers exhibit this signature. These data imply the existence of a family of DNA lesions, arising from both endogenous and exogenous mutagens, present in low numbers per genome but persisting for months to years, that can generate sizable fractions of cells’ mutation burdens.

Background Our randomized phase II trial “DECIDER” showed that the addition of ATRA to decitabine (DAC) significantly improves overall survival in newly diagnosed elderly AML pts (Lübbert et al., J Clin Oncol. 2020). Subgroup analyses revealed that half of the TP53 mutated (MUT) pts responding to DAC+ATRA (DA) lived for >3 years (Bresser et al., Eur J Haematol. 2024, Thomas, Rehman et al., Clin Epigen. 2024). This notable in vivo cooperativity requires elucidation. Methods We employed a MOLM13 isogenic model of TP53+/+, R175H/-, M237I/-, R248Q/-, R273H/-, R282W/-, and TP53-/- clones treated with 100nM DAC by 3 daily pulses, 250nM ATRA on day 4, and studied for proliferation, viability, cell cycle (EdU, Ki67), differentiation (CD11b) at 24h, 48h and 72h post ATRA. RNA-sequencing was performed on TP53+/+ and TP53-/- with results used for gene set enrichment analyses (GSEA) on ‘Hallmark pathways’. Results DA significantly reduced proliferation, induced differentiation and apoptosis in TP53 isogenic model compared to the single agents. The global transcriptomic profile of DA- treated TP53+/+ and TP53-/- cells was distinct from single-agent treatments, as the majority of DEGs were unique to the combination, hence indicating that DAC sensitizes cells to ATRA’s effect. GSEA shows that DA activates the TP53 pathway, as most proliferative pathways were depleted in a p53-dependent manner. Interestingly, ‘Hallmark MYC target genes’ along with MYC transcript, were depleted in both clones, hence pointing towards MYC as an important downstream target for DA, independent of p53. DA treatment significantly reduced MYC+ population in TP53+/+ ( by 48%), R175H/- (55%), M237I/- (25%), R248Q/- (65%), R273H/- (32%), R282W/- (65%), and TP53-/- (25%) clones respectively, as early as 24 hours post ATRA, before any significant cell cycle inhibition (Ki67) was noted. DA increased pMYCT58 and global K48 ubiquitination compared to single agent, pointing towards increased proteasomal degradation. Loss of MYC in cells was followed by loss of Ki67. Heterogeneous and clone-dependent trends towards differentiation or apoptosis were observed in MYC- Ki67- populations. R248Q/- and R282W/- clones were more susceptible to apoptosis, while clones M237I/- and R175H/-, resistant to apoptosis, were susceptible to differentiation. R273H/- clone was vulnerable to apoptosis post differentiation. Conclusions The addition of ATRA to DAC enhances proteasomal degradation of MYC, resulting in reduced proliferation, enhanced apoptosis and increased differentiation in the TP53 isogenic model, with notable differences between the mutated clones studied. Functional association of DA with MYC reduction and validation of primary DECIDER samples with single cell omics is ongoing. Our results support the repurposing of ATRA for treatment of TP53 MUT AML pts. This is pursued in an ongoing phase III, placebo-controlled trial (DECIDER-2, accruing newly diagnosed AML pts with both TP53 WT and TP53 MUT genotypes) with a DAC+VEN backbone and an ATRA vs. placebo randomization. Citation Format: Usama-Ur Rehman, Steffen Boettcher, Michael Lübbert. Sensitization of acute myeloid leukemia cells to the antileukemic activity of all- trans retinoic acid (ATRA) by decitabine is p53-independent [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B020.

Missense mutants of p53, such as the frequent hotspot variant R248Q, exert a dominant-negative effect (DNE) on wild-type (WT) p53 in cancer cells with monoallelic TP53 mutations. However, the precise functional and molecular mechanisms of the DNE have remained elusive due to a lack of appropriate model systems. In this study, we developed a variety of model systems, including CRISPR-edited human isogenic cell lines and transcriptional reporter cell lines, and targeted protein degradation assays that were combined with functional and molecular analyses to functionally characterize the DNE. Formation of heterotetramers between R248Q and WT p53 impaired proper WT p53 functionality by preventing DNA binding and subsequent target gene transactivation. Furthermore, the markedly increased protein half-life of R248Q led to supraphysiologic levels of R248Q, which was critically required for the DNE. Drug-induced targeted protein degradation of R248Q to lower the R248Q:WT ratio restored the transcriptional activity of WT p53, induced antiproliferative effects in cancer cells in vitro, and elicited strong therapeutic activity in vivo. Together, this study provides mechanistic insights into the DNE of p53 missense mutants and indicates that the DNE represents a promising therapeutic target.Significance: Heterotetramerization between R248Q mutant and wild-type p53 in conjunction with supraphysiologic p53R248Q accumulation underlies the dominant-negative effect, highlighting the need to develop pharmacologic strategies to decrease the elevated R248Q:WT ratio.See related commentary by Gencel-Augusto and Lozano, p. 1955

ABSTRACT:TP53-Y220C is a recurrent hot spot mutation in cancers and leukemias. It is observed predominantly in acute myeloid leukemia (AML)/myelodysplastic syndromes among hematological malignancies and is associated with poor outcome. The mutation creates a structural pocket in the p53 protein. PC14586 (rezatapopt) is a small molecule designed to bind to this pocket and thus restore a p53 wild-type (p53-WT) conformation. We demonstrate that PC14586 converts p53-Y220C into a p53-WT conformation and activates p53 transcriptional targets but surprisingly induces limited/no apoptosis in TP53-Y220C AML. Mechanistically, MDM2 induced by PC14586-activated conformational p53-WT and the nuclear exporter exportin 1 (XPO1) reduce the transcriptional activities of p53, which are fully restored by inhibition of MDM2 and/or XPO1. Importantly, p53-WT protein can bind to B-cell lymphoma 2 (BCL-2), competing with BCL-2-associated X protein (BAX) in the BH3 binding pocket of BCL-2, and also binds to BCL-xL and myeloid cell leukemia 1 (MCL-1). However, such binding by PC14586-activated conformational p53-WT is not detected. Pharmacological inhibition of the BCL-2/BAX interaction with venetoclax fully compensates for this deficiency, induces massive cell death in AML cells and stem/progenitor cells in vitro, and prolongs survival of TP53-Y220C AML xenografts in vivo. Collectively, we identified transcription-dependent and -independent mechanisms that limit the apoptogenic activities of reactivated conformational p53-WT and suggest approaches to optimize apoptosis induction in TP53-mutant leukemia. A clinical trial of PC14586 in TP53-Y220C AML/myelodysplastic syndromes has recently been initiated. This trial was registered at www.ClinicalTrials.gov as #NCT06616636.

Receptor tyrosine kinase ERBB4 (HER4) is frequently mutated in human cancer, and ERBB4 mutations have been identified in patients relapsing on targeted therapy. Here, we addressed the functional consequences of recurrent cancer‐associated ERBB4 mutations that are located at regions important for receptor activation and/or are paralogous to known oncogenic hotspot mutations in other ERBB genes. Eleven out of 18 analyzed mutations were transforming in cell models, thus suggesting oncogenic potential for more than half of the recurrent ERBB4 mutations. More detailed analyses of the most potent mutations, S303F, E452K, and L798R, showed that they are activating, can co‐operate with other ERBB receptors and are sensitive to clinically available second‐generation pan‐ERBB inhibitors neratinib, afatinib, and dacomitinib. Furthermore, the S303F mutation, together with a previously identified activating ERBB4 mutation, E715K, promoted resistance to third‐generation EGFR inhibitor osimertinib in EGFR‐mutant lung cancer model in vitro and in vivo. Together, these results are expected to facilitate clinical interpretation of the most recurrent cancer‐associated ERBB4 mutations. The findings provide rationale for testing the efficacy of clinically used pan‐ERBB inhibitors in patients harboring driver ERBB4 mutations both in the treatment‐naïve setting, and upon development of resistance to targeted agents.

MP0533 is a tetra-specific CD3-engaging DARPin designed for avidity-driven T cell-mediated killing of acute myeloid leukemia (AML) cells expressing ≥2 of the 3 leukemia-associated antigens CD33, CD123, and CD70, while sparing healthy cells. MP0533 is evaluated for the treatment of adults with AML or myelodysplastic syndrome (MDS)/AML. The results of the Phase 1/2a dose-escalating dosing regimens (DR) 1–7 showed an acceptable safety profile with evidence of target engagement, T cell activation, and preliminary antitumor activity. However, serum pharmacokinetics (PK) suggested that MP0533 exposure is impacted by target-mediated drug disposition and anti-drug antibodies (ADA) (Jongen-Lavrencic et al. ASH 2024). To mitigate these effects and optimize exposure to treatment, initial MP0533 dose-densification in cycle 1 was introduced in DR 8. Encouraging preliminary antitumor activity was observed (Bories et al. EHA 2025). DR 9 comprises further dose-densification plus anti-CD20 pretreatment. We report the latest results of this first-in-human, multicenter, open-label, Phase 1/2a study of MP0533 (NCT05673057) with focus on the outcomes of DR 8 and 9. Safety, PK, pharmacodynamics (PD), ADA, and antileukemic activity of MP0533 are assessed. Whilst DR 1–7 utilized MP0533 step-up dosing (SUD) on day 1, 5, 8, followed by target dose (TD) on day 15, DR 8 implemented a higher starting dose and earlier TD administration on day 12. Across DR 1–8, TD on day 15 was followed by weekly dosing (28-day cycles). For DR 9, MP0533 is administered with higher frequency vs DR 8 in cycle 1 and beyond, reaching the TD on day 3, and obinutuzumab is given >3 days prior to the first MP0533 dose. Treatment-emergent adverse events (TEAEs) are assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events v5.0. Response is evaluated at weeks 4, 8, and 12 using 2022 European Leukemia Net (ELN) criteria, with additional bone marrow assessment (BMA) on day 14 in DR 9. Centralized molecular measurable residual disease (MRD) is conducted using next-generation sequencing and polymerase chain reaction. As of 21 Jul 2025, 52 patients with relapsed/refractory disease were treated (DR 1=1, DR 2=1; DR 3=3; DR 4=6, DR 5=8, DR 6=9, DR 7=9, DR 8=10, DR 9=5). Median baseline age was 73 years (range 22–82). ELN genetic risk was adverse in 35 patients (67%) and intermediate in 14 (27%). Thirty-one patients (60%) received ≥2 prior treatment lines. Patients received a median of 6 TDs (range 1–21) in DR 1–7 (complete) and 6 TDs (range 1–40) in DR 8 (1 patient still on treatment). DR 9 is ongoing, with a median of 13 TDs (range 8–21) administered; 2 patients completed the 28-day dose-limiting toxicity (DLT) period to date. In addition to the 3 DLTs reported previously (DR7: proctitis, muscular weakness; DR 8: fatal pulmonary hemorrhage in context of disseminated intravascular coagulation, resulting in TD decrease for subsequent patients), 1 DLT of grade 4 liver enzyme elevation was observed in DR 9. The most frequent MP0533-related TEAEs across DR 1–9 were cytokine release syndrome (CRS, 35 patients [67%]) and infusion-related reactions (IRRs, 25 patients [48%]). Three CRS and 6 IRRs transiently reached grade 3 (DR 5–8); all others, including those reported in DR 9, remained of grade ≤2. In DR 8, 3 of 8 evaluable patients achieved a response (1 complete remission [CR]; 2 CR with partial hematologic recovery) vs 4 of 33 in DR 1–7 (1 CR, 3 morphologic leukemia-free state). Two patients in DR 8 maintained response for ≥12 weeks, including 1 in ongoing CR for >9 months. One of the 3 responders in DR 8 showed transient MRD negativity and the 2 others a decrease of variant allele frequency from baseline mutations. In DR 9 to date, 1 of 4 patients with available BMA up to day 14 responded (1 CR) and 2 further showed initial signs of blast count reduction. Serum PK data from DR 8 showed higher drug exposure in cycle 1 vs previous DRs; PK and ADA assessments of DR 9 to confirm expected levels of MP0533 exposure with densified treatment regimen and anti-CD20 pre-treatment is ongoing. MP0533 shows an acceptable safety profile across DR 1–9. Based on initial data, densified MP0533 dosing and anti-CD20 pre-treatment appear tolerable. Preliminary antitumor activity signs with the densified treatment regimens are encouraging. Further efficacy and PK/PD data for DR 9 are currently being collected.

Increased expression of the chemokine CCL2 in tumor cells correlates with enhanced metastasis, poor prognosis, and recruitment of CCR2(+)Ly6C(hi) monocytes. However, the mechanisms driving tumor cell extravasation through the endothelium remain elusive. Here, we describe CCL2 upregulation in metastatic UICC stage IV colon carcinomas and demonstrate that tumor cell-derived CCL2 activates the CCR2(+) endothelium to increase vascular permeability in vivo. CCR2 deficiency prevents colon carcinoma extravasation and metastasis. Of note, CCR2 expression on radio-resistant cells or endothelial CCR2 expression restores extravasation and metastasis in Ccr2(-/-) mice. Reduction of CCR2 expression on myeloid cells decreases but does not prevent metastasis. CCL2-induced vascular permeability and metastasis is dependent on JAK2-Stat5 and p38MAPK signaling. Our study identifies potential targets for treating CCL2-dependent metastasis.

Menin is the scaffolding protein part of the mixed lineage leukemia (MLL) histone methyltransferase complex and uniquely acts as both a tumor suppressor and oncogenic cofactor. KMT2A or MLL rearranged (KMT2Ar) leukemias are a distinct group with poor prognosis and a median overall survival of 0.9 years (Issa, 2021). This interaction has been targeted by menin inhibitors revunemib and ziftomenib with promising results in clinical trials (Aldoss & Issa, 2023; Erba, 2022), and revumenib is now FDA-approved. Menin directly interacts with the TAD domain of c-MYC (MYC) and amplifies MYC’s transcriptional activities (Wu, 2017). We have investigated GT19715, the first-in-class MYC/GSPT1 degrader in MYC-driven hematological malignancies (Nishida, ASH 2022, 2023, 2024). We hypothesized that combined menin inhibition and MYC degradation induces synergistic cytotoxicity in KMT2Ar AML. MOLM-13 CRISPR-generated isogenic cells with wild-type (WT), knockout (KO), or mutant TP53 (R175H, Y220C, M237I, R248Q, R237H, and R282W), MV411 and OCI-AML2 cell lines were exposed to menin inhibitor SNDX-50469 (SNDX) (selleckchem) in combination with GT19715 (GT). Cell death was determined by annexin V/DAPI assays. Time course experiments were conducted in MOLM13 WT, KO, Y220C and R282W cells. Bliss synergy scores (BSS) > 10 are considered synergistic. MOLM-13 cells WT and Y220C mutation labeled with GFP/luciferase were injected into NSG mice and SNDX and/or GT were administered. The combination treatment (100 or 400 nM SNDX and 2 or 4 nM GT) significantly induced cell death and reduced cell numbers by > 99% (> log10^2 difference), starting at day 2 (WT and KO) or 3 (Y220C and R282W) compared to SNDX or GT monotherapy. SNDX + GT induced synergistic cell death (BSS > 10) in all cell lines tested with BSSs up to 80, suggesting substantial synergistic effects by menin inhibition combined with MYC protein degradation. We injected MOLM-13 TP53 WT GFP/Luc and MOLM-13 TP53 Y220C GFP/Luc cell lines (0.5 e6/mouse through tail veins) into NSG mice. After confirming engraftment determined by bioluminescence imaging (BLI), we treated mice with vehicle, GT (3 times a week, every other day by IP injections), SNDX (PO, QD) or GT + SNDX combination for 6 weeks. The combination treatment reduced the total BLI signals at week 2 by 10^4, 10^4 and 10^2 compared to vehicle, GT or SNDX alone. The data suggest the combinatorial treatment of SNDX with GT synergistically reduced leukemia burden in vivo. Combinatorial approach of menin inhibition and MYC degradation results in a profound synergistic reduction of KMT2Ar AML cells in vitro and in vivo, validating the underlying premise. Further mechanistic studies and efficacy in PDX models are ongoing. Shreya Nair, Shayaun Khazaei, Lauren B. Ostermann, Hideaki Mizuno, Po Yee Mak, Bing Z. Carter, Steffen Boettcher, Liandong Ma, Miriam B. Garcia, Branko Cuglievan, Michael Andreeff, Yuki Nishida. KMT2A rearranged acute myeloid leukemia is vulnerable to the combinatorial inhibition of menin and MYC protein degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4306.

KMT2A rearranged (KMT2Ar) leukemias are an aggressive subset of acute leukemias with poor outcomes. Pharmacological inhibition of the menin-KMT2A interaction using revumenib and ziftomenib has shown encouraging efficacy in clinical trials, and revumenib has been FDA-approved (Aldoss & Issa, 2023). However, many patients do not respond to or relapse after menin inhibition as monotherapy. Menin binds directly to the TAD domain of the oncogene MYC, potentiating MYC-mediated transcriptional programs (Wu, 2017). Importantly, PRC1.1 silencing-mediated derepression of MYC mediates menin inhibitor resistance in KMT2Ar AML (Zhou, 2024). Additionally, RAS mutant clones, which are frequently identified in monocytic AMLs with MCL-1 overexpression, are enriched after menin inhibition in patients with KMT2Ar leukemias (Mahdavi, 2024, biorxiv 532874), suggesting an additional resistance mechanism. Recently, we found that MYC regulates transcription of GSPT1, the key protein translation termination factor, and GSPT1 recognizes the stop codon of MYC to promote its translation, forming a feedforward loop. By targeting the MYC-GSPT1 axis, GT19715, the first-in-class MYC/GSPT1 degrader, has shown promising activity in c-MYC-driven hematological malignancies through disruption of MYC/GSPT1 protein translation (Nishida, biorxiv 650490, in revision). We therefore hypothesize that co-targeting of menin and MYC will induce synergistic cytotoxicity in KMT2Ar AML through (1) transcriptional inhibition of MYC by menin inhibition and (2) translation blockade through MYC/GSPT1 degradation. To determine MYC/GSPT1 degradation-mediated inhibition of nascent protein translation, we used O-propargyl-puromycin (OPP) flow-cytometry, which interrogates protein translation at the single-cell level in KMT2Ar AML cells. MOLM-13, MV4;11 and OCI-AML2 cells were treated with menin inhibitor SNDX-50469 (SNDX) and GT19715 (GT). Cell death was determined by Annexin V/DAPI flow cytometry, and time-course studies were performed. Synergy was defined as Bliss scores (BSS) >10. Quantitative PCR was performed to determine mRNA levels. GFP/luciferase-labeled MOLM-13 TP53 WT cells were injected into NSG mice to investigate the in vivo activity of SNDX and GT. Combinatorial treatment of SNDX and GT substantially reduced nascent protein translation determined by OPP incorporation compared to SNDX and GT monotherapy, suggesting the enhanced reduction of nascent protein translation by combined menin inhibition and MYC/GSPT1 protein degradation. The combinatorial treatment induced over 99% cell death in MOLM-13 cells compared to monotherapy, resulting in significantly synergistic cytotoxicity (BSS ~ 80). Mechanistically, SNDX + GT combination reduced c-MYC protein levels compared to monotherapy in a time-dependent manner in MOLM-13 cells. qPCR confirmed marked reductions in MYC mRNA levels after combination treatment in MOLM-13 cells, confirming our hypothesis of inhibiting MYC at transcription and translation. Interestingly, the combination treatment reduced p-ERK and MCL-1 protein levels by more than 90% in MOLM-13 TP53 WT and TP53 Y220C cells before cells underwent apoptosis, suggesting that the combination treatment reduces essential oncogenic proteins in a TP53-independent manner. Interestingly, MCL-1 overexpressing MV4;11 cells were less sensitive to SNDX compared to MV4;11 cells with empty vector control. The combination treatment induced synergistic cell death (BSS > 65) in MCL-1 overexpressing MV4;11 cells, accompanied by a 90% decrease in MCL-1 protein levels. MOLM-13 cells with NRAS G12D showed reduced cell death to SNDX compared to MOLM-13 parental cells, but combinatorial treatment induced synergistic cell death in MOLM-13 cells with NRAS G12D (BSS = 64). The data suggest that the combination treatment overcomes MCL-1 and RAS-mediated resistance to menin inhibition. Finally, combinatorial treatment resulted in a significant reduction of tumor burden in mice injected with MOLM-13 cells. The proposed combinatorial approach of menin inhibition and MYC/GSPT1 protein degradation induces highly synergistic cell death in KMT2A-r AML cells in vitro and in vivo, effectively overcoming resistance by MCL-1 overexpression and RAS pathway activation. Investigations using KMT2Ar AML patient-derived xenograft models are ongoing and will be presented.

Direct targeting of the oncoprotein MYC has long been attempted in cancer therapy, with limited success. We here identify a novel co-regulatory feedback loop of MYC and G1 to S phase transition protein 1 (GSPT1), where MYC promotes transcription of GSPT1, and GSPT1 senses stop codon of MYC to promote its translation. We report on the first-in-class dual MYC/GSPT1 protein degrader, GT19630. GT19630 significantly induced integrated stress response, abrogated oxidative phosphorylation through inhibition of the TCA cycle and induced cell death. Protein degradation of MYC was critical for efficacy of GT19630. GT19630 induced profound anti-proliferative effects and apoptosis agnostic to TP53 in a broad range of cancer cells, and is highly active in vivo in multiple, therapy-resistant hematologic and solid tumor models. Dual MYC/GSPT1 degradation was well tolerated in humanized CrbnI391V mice. In conclusion, we propose a novel treatment approach by directly targeting the MYC-GSPT1 axis in MYC-driven cancers. Statement of significance MYC has been considered an undruggable protein. We found a targetable, novel positive co-regulatory feedback of MYC and GSPT1, a key translation terminator. The dual MYC/GSPT1 degrader GT19630 is highly active in MYC-driven tumors, with moderate effects on humanized Crbn mice, providing opportunities to improve treatment outcome of MYC-driven cancers. ### Competing Interest Statement Declaration of Conflict of Interests: Y.N., Kintor Pharmaceutical: Research Funding. Y.T., Q.Z., Z.Y., H.Y. and D.C. are employees of Kintor Pharmaceutical Ltd. The patent for GT19630 has been filed under WO2022268066A1. A.M., Celgene: Research Funding; Lin BioScience: Research Funding. K.S., Daiichi-Sankyo: Consultancy; Otsuka: Lecture fees; Enliven: Research Funding; Chugai: Lecture fees; Pfizer: Consultancy; Novartis: Consultancy, Research Funding. S.B., Servier: Consultancy; Astellas: Consultancy; Pfizer: Consultancy. T.H., MLL Munich Leukemia Laboratory: Current Employment, Equity Ownership. L.M., Employee of Oncobio Therapeutics, Inc. M.A., Consultancy and Research Funding, Daiichi-Sankyo Inc.; Research Funding, Oxford Biomedical, Eterna Therapeutics Inc., Senti Bio, Sellas, Ellipses Pharma, Kintor Pharmaceutical Ltd., Syndax; Stocks or stock options, Eutropics, SentiBio, Eterna, Chimerix, Oncolyze; Oncobio; Honoraria, Eterna, SentiBio, Syndax, Ona, Sellas, Paraza. There are no other conflicts of interest in all other authors. Paul and Mary Haas Chair in Genetics, , MD Anderson–UT Austin Collaborative Grant, , Cancer Prevention Research Institute of Texas, , RP130397 National Institutes of Health, Cancer Center Support Grant, , P30CA016672 National Institutes of Health , National Cancer Institute, , R21CA267401 The University of Texas MD Anderson Cancer Center MDS and AML Moon Shot, , Kintor Pharmaceutical Research Funding, , TRIUMPH Fellowship in MD Anderson’s CPRIT Research Training Program, , RP210028 National Institutes of Health, , F32CA271697 CURE Childhood Cancer Translation to CURE Award, ,

Therapy-related acute myeloid leukemia and myelodysplastic neoplasms (t-AML/MDS) are devastating complications of chemo- or radiation therapy in patients treated for an unrelated primary malignancy. Cancer patients with TP53-mutant hematopoietic stem and progenitor cells (HSPCs) – a condition termed clonal hematopoiesis (CH) – are at a particularly high risk for t-AML/MDS. However, the pathogenesis of TP53-mutant t-AML/MDS, especially the role of the TP53 allelic state (i.e., mono- vs. biallelic), and its prognostic impact in AML/MDS have remained only poorly understood. We developed novel in vitro and in vivo mouse models to investigate how mono- or biallelic Trp53 mutations influence clonal expansion and leukemic progression from CH to t-AML/MDS. While HSPCs with monoallelic Trp53 mutations gain clonal fitness but retain their genomic integrity under chemo- or radiation therapy, biallelic Trp53 mutations result in genomic instability and are essential for leukemic transformation. Moreover, we provide proof of concept that non-mutational p53 inactivation, such as MDM2 overexpression, can replicate the effects of biallelic TP53 mutations, providing a possible explanation for cases of TP53-mutant AML/MDS that retain one wild-type TP53 allele. Our findings elucidate the pathogenesis of TP53-mutant t-AML/MDS and support the classification of biallelic TP53-mutant AML/MDS as distinct clinical entities.

Acute myeloid leukemia with mutations in TP53 (TP53mut AML) is fatal with a median survival of 6 months. RNA sequencing on purified AML patient samples showed that TP53mut AML had higher expression of mevalonate pathway genes. Using novel, isogenic TP53mut AML cell lines and primary samples, we determined that TP53mut AML resistance to AML chemotherapy cytarabine (AraC) correlated with increased mevalonate pathway activity, a lower induction of reactive oxygen species (ROS), and a mitochondrial response with increased mitochondrial mass and oxidative phosphorylation. Pretreatment with the statin class of mevalonate pathway inhibitors reversed these effects and chemosensitized TP53mut AML. The geranylgeranyl pyrophosphate (GGPP) branch of the mevalonate pathway was required for TP53mut AML chemoresistance. In addition to its role in mitochondria biogenesis, we identified a novel function of GGPP in regulating glutathione for management of AraC-induced ROS. However, statins alone were inadequate to fully reverse chemoresistance in vivo and in a retrospective study of 364 TP53mut AML patients who received chemotherapy concurrently with a statin. Finally, we identified clinical settings and strategies to successfully target the mevalonate pathway, particularly to address the unmet need of TP53mut AML.

Tumor protein 53 (TP53)-mutated acute myeloid leukemia (AML) is characterized by poor outcomes and the quick development of treatment resistance. Here, we report that simultaneous inhibition of cyclin-dependent kinases (CDKs) and histone deacetylases (HDACs) with dinaciclib and CAY10603, respectively, eliminates the therapeutic response gap between TP53-mutant and TP53 wild-type AML. Biochemical profiling showed that CAY10603 is not only HDAC6-selective but also exhibits pan-HDAC activity similar to suberoylanilide hydroxamic acid, enabling dual targeting of transcriptional and cell cycle pathways. Across parental wild-type lines and isogenic TP53 mutants, the combination consistently suppressed clonogenic growth, induced caspase-dependent apoptosis, and downregulated key regulators such as CDK2, CDK4/6, and their cyclins, while restoring the CDK inhibitor CDKN1A/p21. In an orthotopic NSG mouse model, dinaciclib + CAY10603 significantly reduced leukemia burden and extended survival without adverse toxicity. By “normalizing” TP53-mutant AML to respond like its wild-type counterpart, this pan-HDAC/multi-CDK blockade offers a TP53-agnostic therapeutic option and warrants clinical evaluation as a strategy that remains effective regardless of baseline allelic status.