TP53-mutated Acute Myeloid Leukemia Research Articles

P53 immunohistochemistry as an ancillary tool for rapid assessment of residual disease in TP53-mutated acute myeloid leukemia and myelodysplastic syndromes.

Measurable residual disease flow cytometry (MRD-FC) and molecular studies are the most sensitive methods for detecting residual malignant populations after therapy for TP53-mutated acute myeloid leukemia and myelodysplastic neoplasms (TP53+ AML/MDS). However, their sensitivity is limited in suboptimal aspirates or when the immunophenotype of the neoplastic blasts overlaps with erythroids or normal maturing myeloid cells. In this study, we set out to determine if p53 immunohistochemistry (IHC) correlates with MRD-FC and next-generation sequencing (NGS) in the posttherapy setting and to determine the utility of p53 IHC to detect residual disease in the setting of negative or equivocal MRD-FC. We retrospectively identified 28 pre- and posttherapy bone marrow biopsy specimens from 9 patients with TP53+ AML/MDS and a p53 overexpressor phenotype by IHC (strong 3+ staining at initial diagnosis). Next-generation sequencing and/or MRD-FC results were collected for each specimen. Using a threshold of more than ten 2-3+ cells in any one 400× field, p53 IHC detected residual disease with a sensitivity of 94% and a specificity of 89%. The threshold used in this study showed a high degree of concordance among 6 blinded pathologists (Fleiss κ = 0.97). Our study suggests that p53 IHC can be used as a rapid tool (within 24 hours) to aid in the detection of residual disease that may complement MRD-FC or NGS in cases in which the flow cytometry immunophenotype is equivocal and/or the bone marrow aspirate is suboptimal.

Molecular ontogeny underlies the benefit of adding venetoclax to hypomethylating agents in newly diagnosed AML patients.

The clinical impact of molecular ontogeny in acute myeloid leukemia (AML) was defined in patients treated with intensive chemotherapy. In a cohort of 314 newly diagnosed AML patients, we evaluated whether molecular ontogeny subgroups have differential benefit of venetoclax (VEN) added to hypomethylating agents (HMA). In secondary ontogeny (n = 115), median overall survival (OS)(14.1 vs. 6.9 months, P = 0.0054), composite complete remission (cCR 61% vs. 18%, P < 0.001) and allogeneic hematopoietic stem cell transplant (alloHCT) (24% vs. 6%, P = 0.02) rates were better in patients treated with HMA + VEN vs. HMA. In contrast, in TP53 AML(n = 111) median OS (5.7 vs. 6.1, P = 0.93), cCR (33% vs. 37%, P = 0.82) and alloHCT rates (15% vs. 8%, P = 0.38) did not differ between HMA + VEN vs. HMA. The benefit of VEN addition in the secondary group was preserved after adjustment for significant clinicopathologic variables (HR 0.59 [95% CI 0.38-0.94], P = 0.025). The OS benefit of HMA + VEN in secondary ontogeny was similar in those with vs. without splicing mutations (P = 0.92). Secondary ontogeny AML highlights a group of patients whose disease is selectively responsive to VEN added to HMA and that the addition of VEN has no clinical benefit in TP53-mutated AML.

Abstract 5589: CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine

Abstract CBFB::MYH11 acute myeloid leukemia (AML) is a subset of Core Binding Factor AML initiated by an oncofusion in which Core Binding Factor Beta (CBFB) is fused to the C-terminus of smooth muscle myosin heavy chain 11 (MYH11). CBFB::MYH11 AML has a high cure rate with conventional anthracycline/cytarabine induction and high-dose cytarabine consolidation in patients under the age of 60, and remains chemosensitive after multiple relapses; the mechanisms underlying chemosensitivity are unknown. TP53 is a key regulator of DNA damage response and apoptosis, and TP53-mutated AML is often chemoresistant. Large sequencing studies have revealed only rare co-occurrence of TP53 mutations with CBFB::MYH11. We initially used CBFB::MYH11 as a negative control for an unrelated study of TP53 function and unexpectedly found striking synergy. While expression of CBFB::MYH11 in WT murine hematopoietic stem and progenitor cells (HSPCs) induced a low-penetrance (~20%), long latency (~6 months) myelomonocytic AML, expression of CBFB::MYH11 in Trp53-/- HSPCs induced AML with 100% penetrance and a median latency of 8 weeks. CBFB::MYH11-induced AMLs arising in Trp53-/- cells were similar by morphology and flow cytometry phenotype. We validated these findings in a second model, using a conditional Trp53flox/flox x Vav1-Cre model in which Trp53 is inactivated in HSPCs, with nearly identical results. Trp53-/- cells transduced with Empty Vector did not develop AML, but did develop T cell lymphoma with a median latency of 16 weeks, similar to prior studies. Trp53 deficiency also cooperated with RUNX1::RUNX1T1, the other Core Binding Factor oncofusion, to induce a high-penetrance (100%) and short latency AML (median latency 10 weeks); consistent with prior data, RUNX1::RUNX1T1 expression alone in Trp53 WT cells was insufficient to induce AML. To determine whether Trp53 influences the chemosensitivity of CBFB::MYH11 expressing cells, we performed cytarabine dose-response experiments. While CBFB::MYH11-transduced WT HSPCs were very sensitive to cytarabine (relative to EV), sensitivity was reduced in CBFB::MYH11-transduced Trp53-/- cells, suggesting that CBFB::MYH11-induced chemosensitivity is, at least in part, mediated by the TP53 pathway. To determine whether CBFB::MYH11 expression results in upregulation of key TP53 pathway genes, we transduced WT HSPCs with EV or CBFB::MYH11, transplanted cells into WT mice, harvested bone marrow at 3 months, and performed RNA-Seq. Key TP53 pathway genes were upregulated, including Bbc3/Puma, Bax, Cdkn1a/p21, and Trp53 (all 2.6-4.6-fold, p≤0.05). These data suggest that the chemosensitivity and the favorable prognosis of CBFB::MYH11 AML may be due to the direct activation of the TP53 pathway by CBFB::MYH11. Similar studies are underway with RUNX1::RUNX1T1-transduced cells to determine whether TP53 is relevant for the chemosensitivity of that CBF AML as well. Citation Format: Ryan B. Day, Julia A. Hickman, Casey D. Katerndahl, Sai Mukund Ramakrishnan, Christopher A. Miller, Timothy J. Ley. CBFB::MYH11 expression activates Trp53 in myeloid cells, which inhibits cell growth and sensitizes cells to cytarabine [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 5589.

Abstract 4664: Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting cell cycle checkpoint

Abstract Acute myeloid leukemia (AML) is a hematological malignancy characterized by various somatic mutations leading to abnormal proliferation and differentiation of hematopoietic cells. Mutations on the TP53 gene occur in 5% to 10% of patients with de novo AML, with higher frequency in patients with relapsed and refractory (R/R) AML. Patients with TP53-mutated AML typically exhibit a poor response to conventional induction therapy and have a short overall survival (OS) rate, with a median duration of 5-9 months. Although various target agents have been developed for the treatment of AML, treatment options for TP53-mutated AML remain limited. Here, we have discovered a novel drug combination that can enhance the efficacy of the conventional therapeutic regimen for TP53-mutated AML patients. For in vitro analysis, we used the human AML cell line Kasumi-1(AML-M2), which harbors a homozygous TP53 R248Q mutation. We treated kasumi-1 cells with a combination of cytarabine and Idarubicin to produce drug tolerant persisters (DTPs) which is a minor population that survived the drug treatment. The Kasumi-1 DTPs displayed temporal G2/M phase cell cycle arrest and significant resistance to further repeated drug treatment. To discover drug candidates targeting TP53-mutated DTPs, we first examined the differentially expressed genes (DEGs) of TP53-mutated AML patients using patients’ RNA expression data deposited in the cancer genome atlas (TCGA) database. Using selected DEGs, drug candidates potentially affecting TP53-mutated AML cells were extracted from the connectivity map (CMap build 02) database. In addition, through a literature search, drugs with the ability to reactivate mutant p53, drugs that directly target mutant p53, synthetic lethal partners with mutant p53, and drugs that target leukemia stem cells were selected as candidate groups. As a result, we selected 46 drug candidates that would be effective for TP53-mutated AML through in silico analysis and therapy strategies targeting p53. Using in vitro cytotoxicity assay, we screened 46 candidate compounds at multiple doses and confirmed several drugs that effectively eliminate DTPs. The drug screening showed that cell cycle checkpoint inhibitors not only resulted in a significant reduction of G2/M phase arrested cells but also induction of apoptotic cells at the subG1 phase. Furthermore, the zero interaction potency (ZIP) model of SynergyFinder (https://synergyfinder.fimm.fi) analysis showed that cell cycle checkpoint inhibitors synergistically enhanced the cytotoxic effect of cytarabine on Kasumi-1 DTPs. Collectively, this study suggests that novel combination treatment of cell cycle checkpoint inhibitors with conventional drugs selectively inhibits TP53-mutated AML DTPs. Citation Format: Daehyeon Gwak, Dongchan Kim, Heejun Jang, Jun Liu, Suji Min, Ja Min Byun, Youngil Koh, Junshik Hong, Sung-soo Yoon, Dong-Yeop Shin, Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea, Republic of. Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting cell cycle checkpoint [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 4664.

Myelodysplasia-related gene mutations are associated with favorable prognosis in patients with TP53-mutant acute myeloid leukemia.

This study aimed to examine the characteristics and treatment outcomes of patients with TP53-mutant acute myeloid leukaemia (AML) and to explore potential prognostic factors. This retrospective analysis included 130 patients diagnosed with TP53-mutant AML at the Fujian Medical University Union Hospital between January 2016 and June 2023. Patients' ages ranged from 17 to 80 years, with a median age of 59 years. The proportions of de novo, therapy-related, and secondary AML cases were 71.5%, 7.7%, and 20.8%, respectively. Complex karyotypes were observed in 60.6% of patients, and the proportions of -5 or del(5q), -7 or del(7q), and - 17 or del(17p) were 41.7%, 27.9% and 14.4%, respectively. DNA methylation- and myelodysplasia-related (MR) gene mutations were observed in 36.9% and 25.4% of patients, respectively. These patients showed poor survival, with a median overall survival (OS) of 4.5 months, a 1-year OS rate of 32.5%, a 3-year OS rate of 18.8%, and a 5-year OS rate of 11.3%. The complete response rates for intensive chemotherapy (IC), hypomethylating agent (HMAs)-based therapies, and azacitidine plus venetoclax were 35.7%, 22.2%, and 37.5%, respectively. Patients who did or did not receive allogeneic haematopoietic stem cell transplantation (allo-HSCT) had similar prognoses (median OS: 6.0 vs. 3.9 months; P = 0.6415). Multivariate analysis indicated that MR gene mutations is an independent favorable prognostic factor of OS (HR = 0.366, 95% CI: 0.181-0.738, P = 0.005). In conclusion, patients with TP53-mutant AML have poor prognoses under current treatment strategies and MR gene mutations are associated with a more favorable survival. Therefore, further studies are needed to improve the survival rates in this population.

Targeting the mevalonate or Wnt pathways to overcome CAR T-cell resistance in TP53-mutant AML cells

TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are characterized by chemotherapy resistance and represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cells might be a promising therapeutic option for TP53-mutant AML/MDS. However, the impact of TP53 deficiency in AML cells on the efficacy of CAR T-cells is unknown. We here show that CAR T-cells engaging TP53-deficient leukemia cells exhibit a prolonged interaction time, upregulate exhaustion markers, and are inefficient to control AML cell outgrowth in vitro and in vivo compared to TP53 wild-type cells. Transcriptional profiling revealed that the mevalonate pathway is upregulated in TP53-deficient AML cells under CAR T-cell attack, while CAR T-cells engaging TP53-deficient AML cells downregulate the Wnt pathway. In vitro rational targeting of either of these pathways rescues AML cell sensitivity to CAR T-cell-mediated killing. We thus demonstrate that TP53 deficiency confers resistance to CAR T-cell therapy and identify the mevalonate pathway as a therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising CAR T-cell therapy-enhancing approach for TP53-deficient AML/MDS.

Unmet Horizons: Assessing the Challenges in the Treatment of TP53-Mutated Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) remains a challenging hematologic malignancy. The presence of TP53 mutations in AML poses a therapeutic challenge, considering that standard treatments face significant setbacks in achieving meaningful responses. There is a pressing need for the development of innovative treatment modalities to overcome resistance to conventional treatments attributable to the unique biology of TP53-mutated (TP53mut) AML. This review underscores the role of TP53 mutations in AML, examines the current landscape of treatment options, and highlights novel therapeutic approaches, including targeted therapies, combination regimens, and emerging immunotherapies, as well as agents being explored in preclinical studies according to their potential to address the unique hurdles posed by TP53mut AML.

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.

TP53-Mutated Acute Myeloid Leukemia Patients Treated with Intensive Therapies Have Superior Outcomes: A Single Institution, Retrospective Study

Background: Mutations in TP53, found in 5-20% of acute myeloid leukemia (AML), confer poor prognosis and are associated with high rates of therapy resistance, high relapse rates, and dismal overall survival (OS). Recent advances have provided a variety of options for up-front AML therapy including a liposomal preparation of standard-of-care chemotherapy (CPX-351) and venetoclax-based combinations. Due to known poor outcomes with intensive chemotherapy, many providers are favoring less intense strategies for patients with TP53 mutant AML, but these approaches have not been directly compared for this patient subgroup. Strategies for treating TP53 mutant AML are highly heterogeneous and optimal treatment strategies have not been defined in this setting. We performed a retrospective study to compare the clinical outcomes of TP53 mutant AML patients treated at our center. Methods: The study was a single-center, retrospective cohort analysis conducted at the University of Minnesota. Adult patients (age ≥18 years) with the diagnosis of AML or myeloid sarcoma between January 1, 2014 and July 31, 2022 who have next-generation sequencing panel confirming a pathogenic TP53 mutation and received front-line therapy for AML at our center were included in this study. Kaplan-Meier analysis was used to estimate the probabilities of OS, defined as the date of treatment initiation to the date of death from any cause or last follow-up. Results: Our study includes 88 patients with TP53 mutant AML with available clinical data in an AML registry at our institution. Baseline demographic and clinical characteristics are summarized in Table 1. The median age at diagnosis was 67 years (range 28-87) with male predominance (65.9%). Among this, 15/88 patients (17.1%) had therapy-related AML and 40/88 patients (45.5%) had secondary AML. The median bone marrow blast percentage at diagnosis was 31% and peripheral blast percentage was 9%. The majority of patients have high variant allelic frequency (VAF, 71.9% with VAF &amp;gt; 0.4 for patients with available VAF data) and ASXL1 was the most frequent co-mutation identified followed by KRAS and NRAS. Treatment regimens were classified as intensive or nonintensive. Intensive treatment regimens included infusional cytarabine with either daunorubicin or idarubicin (7+3), liposomal cytarabine and daunorubicin (CPX-351), and mitoxantrone, etoposide and cytarabine (MEC). Nonintensive regimens included hypomethylating agents (decitabine or azacitidine) with or without venetoclax and clofarabine with low-dose subcutaneous cytarabine. A total of 66 patients were included in the survival analysis. There was no significant difference in OS between patients aged &amp;lt; 65 years and ≥ 65 years at diagnosis (2-year OS, 11% versus 5%, P=0.76). Superior 2-year OS was observed in patients who received front-line intensive (n = 31) versus nonintensive (n = 35) induction chemotherapy (2-year OS, 13% versus 3%, P=0.01, Figure 1). Patients who received intensive therapy for any line of treatment also demonstrated superior OS compared to those who never received intensive therapy (2-year OS, 12% versus 3%, P=0.02). The rate of achieving complete remission (CR) was higher in patients who received intensive treatment compared to non-intensive therapy in the first-line (51.6% versus 25.7%, P=0.03). Rate of CR at any time was also higher for patients who received intensive treatment for any line of treatment (50% versus 18.2%, P&amp;lt;0.01). Among the intensive first-line treatment group, 7+3 (n = 18) showed superior OS relative to CPX-351 (n = 12) (2-year OS, 22% versus 0%, P=0.03) although there was no significant difference in CR rate (55.6% versus 50%, P=0.77). Furthermore, significant survival benefit was shown in patients who underwent allogeneic hematopoietic cell transplantation compared to patients who had not (2-year OS, 21% versus 4%, P&amp;lt;0.01]). Conclusions: In our cohort, TP53 mutant AML patients treated with intensive chemotherapy and transplant have superior outcomes to those who do not. Since this was a retrospective study, we cannot rule out the possibility that these results are confounded by providers favoring more intensive therapies for fitter patients. However, these data imply that TP53 mutant patients may benefit from more intensive therapy and suggests that future work to define optimal therapy regimens in this patient population may incorporate intensive strategies.

Post-Transplant T Cell Clonotype Diversity Is Associated with Survival in Patients with TP53-Mutated Acute Myeloid Leukemia

Background TP53 mutations are associated with unfavorable outcomes in various cancer types and present an obstacle to achieving sustained remission in acute myeloid leukemia (AML). Even with allogeneic hematopoietic stem cell transplantation (HSCT), the risk of relapse approximates 80%, with dismal long-term survival. This could imply methods of immune escape in TP53 mutated leukemias that allow them to evade the graft-versus-leukemia (GVL) effect, which is a critical mechanism of disease control with HSCT. This study investigates the dynamics of immune reconstitution at single-cell resolution in TP53-mutated AML at pre- and multiple post-HSCT timepoints in patients who either remain in long-term remission or relapse following HSCT. Methods We collected 26 longitudinal bone marrow aspirates and 1 peripheral blood sample from 12 patients with TP53-mutated AML who underwent HSCT at the Dana-Farber Cancer Institute between 2015-21. All patients received HLA-matched T-cell replete HSCT in morphologic remission, with standard tacrolimus/methotrexate-based prophylaxis and peripheral blood stem cells. We categorized the patients into two cohorts based on their treatment response: long-term remission (&amp;gt;3.5 years, cohort 1, n=4) or relapse (cohort 2, n=8). We performed paired 5' single-cell RNA and T cell receptor (TCR) sequencing on sorted viable mononuclear from each sample and CD3+ T cells from selected samples using 10x Genomics procedures. In total, we analyzed 44 samples (27 mononuclear, 17 CD3+), resulting in 250,640 high-quality single-cell transcriptomes, including 137,649 T cells and NK cells. Data analysis was performed using Cell Ranger Multi (10x Genomics) and R 4.3 with the tidyverse 2.0, Seurat 5.1, and scRepertoire 1.1 packages. Results We focused our initial analysis on the 2-6-month post-HSCT period that is critical for immune reconstitution. This included eight samples from seven patients in the two cohorts (1: remission, 2: relapse, pink box in Figure 1). Of these 7 patients, 4 were in the relapse cohort, with these relapses occurring &amp;gt;4 months post-HSCT. No significant difference in T-cell chimerism was seen between these 7 patients in this 2-6 month period. Based on canonical gene expression signatures, we annotated cell types and selected T cells for further evaluation (Figure 2). First, we found that the CD4/CD8 ratio differed between the cohorts, indicating a relative CD8+ T cell expansion in the relapse cohort (cohort 1: 1.69, cohort 2: 0.55, P&amp;lt;0.0001). Second, we found lower TCR diversity in the relapse cohort (inverse Simpson index, cohort 1: 234.6, cohort 2: 83.0, P&amp;lt;0.0001). This difference remained significant when subsampling the same number of T cells per patient and when restricting all samples to the 3 to 4-month timepoint. Considering the disparity in CD8 T cell numbers between the cohorts, we also analyzed repertoire diversity, specifically in CD8+ T cells, and found that the observed significance persisted. To further investigate TCR diversity, we overlaid T cell clonotypes (cells that share the same TCR sequence) in a UMAP visualization, which highlighted increased hyperexpanded CD8+ T cells in the relapse cohort (Figure 2). Finally, we found that greater clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PRF1, NKG7, and GZMA. These results suggest that cytotoxic CD8+ T cells link lower TCR diversity to the likelihood of disease relapse. Conclusions By performing deep analysis of single-cell gene expression linked to TCR clonotypes, we discover new insights into the biology of hematopoietic reconstitution after HSCT of patients with TP53-mutated AML. Despite having analyzed a limited number of samples, we find that patients who remain in long-term remission have significantly higher CD8+ TCR diversity between 2-6 months post-HSCT, while those who relapsed between 5-18 months post-HSCT showed lower TCR diversity and hyperexpanded CD8+ clones at the same timepoints. This work suggests that early post-HSCT TCR diversity could be a biomarker associated with GVL and warrants further study. In ongoing and future analyses, we will mine the current dataset for further insights into the factors influencing survival in TP53-mutated AML patients. We also plan to extend the measurement of TCR diversity as a predictor of survival for validation in a larger cohort.

The XPO7/Npat Axis Inactivation Is a Therapeutic Vulnerability for TP53-Mutated AML

In acute myeloid leukemia (AML), TP53 mutations are the most prognostically unfavorable genetic alterations, as they confer resistance to various therapeutic agents and form relapsing clones, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes or pathways whose loss is vulnerable to TP53 deficiency in AML cells, we performed genome-wide CRISPR-Cas9 screens using Trp53-knockout (KO) and wild-type (WT) mouse AML cells. We employed two distinct KO screen libraries, through which we identified Xpo7, a putative nuclear/cytoplasmic transporter, as a common factor necessary for Trp53-KO AML survival. Interestingly, Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. Further probing this phenomenon, we performed a CRISPR-Cas9 saturation mutagenesis scan on all Xpo7 exons. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT AMLs. In stark contrast, these same sgRNAs were mostly depleted in Trp53-KO AMLs. To determine the role of Xpo7 depletion on the expansion of Trp53 deficient clones, we prepared AML cells containing a small fraction of Trp53-KO cells both in the Xpo7-WT and KO backgrounds and assessed the proportion over time in vitro or in vivo. Remarkably, the expansion of Trp53-KO clones, noticeable in the Xpo7-WT setting, was significantly reduced under the Xpo7-KO background, prolonging the mice's survival. These data suggest that Xpo7 depletion effectively impedes Trp53-KO clone expansion. Since Xpo7 is presumed to mediate the nuclear import and export of proteins, we hypothesized that Xpo7 contributes to retaining Trp53 protein within the nucleus in Trp53-WT AMLs. As expected, nuclear Trp53 protein and mRNA levels of Trp53-targeting genes decreased significantly upon Xpo7 depletion. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AMLs. Next, to elucidate the underlying mechanisms causing the toxic effects in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose localization was potentially regulated by Xpo7 in the Trp53-KO state. Employing mass spectrometry on an immunoprecipitate using anti-Xpo7 and cytoplasmic/nuclear protein extracts from both Xpo7-WT and KO AML cells, we found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and showed a significant shift towards cytoplasmic localization upon Xpo7 depletion in Trp53-KO AMLs. Strikingly, Npat protein levels displayed a notable upregulation by Trp53 depletion in AML cells. To evaluate the relevance of the XPO7/NPAT axis in human TP53-mutated AML cells, we examined the correlation between XPO7 and NPAT expression levels and AML subtypes and genetic background using publicly available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets. Moreover, XPO7 and NPAT mRNAs levels were remarkably high in acute erythroid leukemia (AEL) cases, where TP53 mutations are frequently observed (Tyner et al. Nature, 2018; Iacobucci et al. Nat Genet. 2019). The Xpo7 and Npat protein expression evaluation using primary AML samples corroborated these findings, revealing overexpression in TP53-mutated AML cases. Finally, we assessed the therapeutic effect of Npat depletion in Trp53-deficient AML cells, using the dTAG system to induce Npat protein degradation by knock-in FKBP12 F36V into Npat loci. dTAG v-1 treatment immediately degraded endogenous Npat protein and led to delayed G1/S transition progression. Moreover, Npat degradation induced DNA damage, particularly in the presence of a WEE1 inhibitor or etoposide, and subsequent apoptosis. Transplantation of these AML cells into immunocompromised mice followed by dTAGv1 treatment for one week demonstrated suppressing leukemia progression in vivo. Furthermore, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed the proliferation of HEL, a human AEL cell line harboring TP53 mutation, in vitro and in vivo. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. We unveiled the novel regulatory mechanism of protein localization by XPO7, depending on TP53 mutational state. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML.

Targeting Venetoclax Resistance in TP53-Mutated Acute Myeloid Leukemia

Introduction: The combination therapy of Venetoclax (Ven) and Azacitidine (Aza) has shown remarkable improvements in the treatment of acute myeloid leukemia (AML) patients not eligible for chemotherapy. Nevertheless, patients with TP53-mutated AML are more prone to have a refractory disease or develop rapid resistance. A recent study indicated that a combined expression ratio of BCL-2, BCL-XL and MCL-1 proteins in leukemic stem cells predicts clinical response to Ven+Aza (Waclawiczek et al. 2023). Furthermore, our previous work has demonstrated that erythroid and megakaryoblastic leukemia blasts are more dependent on BCL-XL than BCL-2 (Kuusanmäki et al. 2023). Emerging evidence suggests that the erythroid-biased differentiation and gene signature is enhanced in many TP53-mutated patients (Rodriquez-Meira et al. 2022). Here we hypothesized that in TP53 -mutated AML, leukemic blasts correspond more closely to megakaryocyte-erythroid progenitors (MEPs) leading to an unfavorable BCL-2 family expression ratio, which can be associated with poor responses to Ven. Consequently, we aimed to identify novel compounds targeting this specific patient subgroup. Methods: We utilized flow cytometry to analyze the mean fluorescent intensity (MFI) of BCL-2 family proteins (BCL-2, BCL-XL, MCL-1) in the CD34+ blast cells of 16 bone marrow-derived AML samples taken before Ven+Aza therapy ( TP53mut n=8, TP53wt n=8). Ex vivo drug testing was performed with eight compounds and blast-specific drug responses were analyzed after 48 hours with flow cytometry. RNA sequencing was performed on CD34+ or CD117+ cells enriched from 35 pre-Ven+Aza treatment samples. To assess erythroid signature scores, we calculated the mean expression of six different erythroid genes ( GATA1, KLF1, ZFPM1, GATA2, GYPA, TFRC) for each patient, following the approach by Rodriquez-Meira et al. 2022. The expression levels of 19 intracellular proteins, including BCL-2, BCL-XL, MCL-1, were quantified from the corresponding cell population of the samples by mass cytometry (CyTOF). Results: In the comparison of BCL-2 family protein expression and treatment responses, BCL-XL levels were significantly higher (p=0.03) in the treatment-resistant group, while no differences were found in BCL-2 or MCL-1 ( Figure 1A). In the TP53-mutated blasts, a trend of decreased BCL-2 was identified compared to the TP53 wild-typeblasts (p=0.08), but no difference in BCL-XL or MCL-1 levels was noted. However, all three Ven+Aza-refractory TP53-mutants exhibited high BCL-XL expression compared to the treatment-responsive TP53-mutated patients. Next, we assessed whether an erythroid-like phenotype was associated with TP53 mutation status by computing the erythroid gene signature scores from the RNAseq data of enriched blasts. Erythroid scores were increased among TP53-mutated samples (p=0.04), and interestingly, a higher erythroid score was linked to lower BCL-2 protein expression, regardless of TP53 mutation status ( Figure 1B). Finally, we assessed the drug efficacy of eight pre-selected compounds, including BCL-2 family inhibitors. As expected, Ven was ineffective in the TP53-mutated samples. In contrast, the TP53-mutated blasts showed higher ex vivo sensitivity to Navitoclax (BCL-2/BCL-XL inhibitor) (Nav IC50=90nM, Ven IC50&amp;gt;1000nM). Additionally, LCL-161 (IAPs inhibitor) demonstrated notable efficacy in the TP53-mutants and was significantly more effective compared to the wild-type controls (LCL-161 IC50=300nM, Ven IC50&amp;gt;1000nM). Discussion: Here, we show that increased BCL-XL protein expression is associated with clinical resistance to Ven+Aza. Furthermore, the link between erythroid gene signature and BCL-2 expression suggests that especially those AML blasts harboring TP53 mutations might have impaired BCL-2 expression due to a closer resemblance to MEPs. The increased sensitivity of TP53-mutated AML cells to LCL-161 demonstrated in the study, highlights the potential of targeting extrinsic apoptosis pathway via inhibiting the inhibitors of apoptosis proteins (IAPs). Furthermore, co-targeting of BCL-2/BCL-XL might provide alternative therapeutic approach in Ven resistant TP53-mutated patients. These findings are currently being validated with a larger patient cohort.

Comparable Survival of Treatment Naïve TP53 Mutated Acute Myeloid Leukemia Treated with Hypomethylating Agent Compared to Hypomethylating Agent Plus Venetoclax Based Therapy

Background TP53-mutated (m) acute myeloid leukemia (AML) is an adverse risk disease, which continues to have dismal outcome in the contemporary era of novel therapies. Venetoclax (VEN) in combination with hypomethylating agents (HMA; azacitidine or decitabine) have shown to improve complete remission (CR) rates compared to HMA alone, however no significant differences in overall survival (OS) have been observed. Methods We conducted a retrospective study through the COMMAND consortium (a collaboration of acute leukemia experts from 10 US academic institutions) to analyze the impact on survival outcomes of HMA plus (+) VEN compared to HMA-based therapies in adult (≥18 years) pts with TP53m AML. The AML Database of 381 pts with TP53m AML diagnosed between 2012-2022 was queried and 154 (40%) (HMA [n=50] and HMA +VEN [n=104]) pts were identified eligible for this analysis. Continuous variables were summarized as median (range) while categorical variables were reported as frequency (percentage). The Kaplan-Meier method was used to estimate OS, defined as time from diagnosis to death or last follow-up. Logistic regression models were used to determine the univariate and multivariate of OS. Multivariable models included all significant univariate predictors. All tests were two-sided with a p value &amp;lt;0.05 considered statistically significant. Results Baseline characteristics and responses The median age (years [yrs]) was 74 (range [R], 38-87) and 71 (R,29-88) yrs in the HMAand HMA + VEN groups (gps) (p= 0.23), respectively ( Table 1). Thirty-four (68%) and 57 (55%) patients (pts) were ≥ 70 yrs in the HMA and HMA + VEN gps, respectively (p= 0.16). Twenty-six percent vs 31% ( p=0.57), 86% vs 90% ( p=0.58) and 68% vs 73% ( p=0.56) of pts had secondary AML, had complex cytogenetics (CG), and had multi-hit TP53m in the HMA and HMA + VEN gps, respectively. The proportion of pts with most frequently occurring (&amp;gt; 5%) myeloid co-mutations ( RUNX1, ASXL1, TET2, DNMT3A, RAS, PTPN11 and JAK2) were not significantly (NS) different between the HMA and HMA + VEN gps, as shown in Table 1. The early treatment related mortality in first 30 days of induction was higher in the HMA+VEN gp (25%) when compared with the HMA gp (6%), p=0.004. The complete remission rates (CR/CRi) were higher in the HMA + VEN gp compared to the HMA gp (35% vs 18%, p=0.05). Similarly, the proportion of pts receiving allogeneic stem cell transplant (allo-HCT) after induction was higher in the HMA + VEN gp when compared with the HMA gp (13% vs 4%; p=0.14). Duration of response and overall survival The median follow up was 9.8 months (mo) (R, 1-56) and 5.80 mo (R, 1-30.4) in the HMA and HMA + VEN gps, respectively ( p=0.12). The median duration of response (DOR) was higher in the HMA +VEN gp (15.6 mo) when compared with the HMA gp (7.93 mo), but NS ( p=0.26). The median OS was NS different when comparing the HMA and HMA + VEN gps (9.23 vs 7.3 mo, p=0.80 [ Figure 1]). Multivariable analysis for duration of response and overall survival We did multivariable analysis (MVA) for DOR and OS using baseline variables ( Table 1) that showed significance/trend towards significance in univariate analysis ( p&amp;lt;0.1). Allo-HCT (HR; 0.11, 95% CI: 0.02-0.52, p=0.005) retained significance for better DOR, whereas age ≥ 70 yrs (HR; 1.7, 95% CI: 0.63-4.65, p=0.28) and complex CG (HR; 3.05, 95% CI: 0.79-11.73, p=0.10) did not retain significance in MVA. In MVA for OS, achievement of CR/CRi (HR; 0.35, 95% CI; 0.20-0.59, p=&amp;lt;0.001) and allo-HCT after induction (HR; 0.06, 95% CI; 0.09-0.46, p=0.007) retained favorable significance for OS, whereas complex CG (HR; 2.36, 95% CI: 1.11-4.99, p=0.02) retained significance for shorter OS. PTPN11m (HR; 1.57, 95%CI: 0.75-3.28, p=0.23) and TET2m (HR; 1.02, 95% CI: 0.56-1.87, p= 0.92) did not retain significance in MVA. Conclusion In this multi-center real-world study, while HMA+VEN led to improvement in CR rates and a higher proportion of pts were bridged to allo-HCT, it did not associate with an improvement in OS when compared with HMA monotherapy. More effective treatment strategies are warranted to improve outcome of pts with TP53m AML.

Safety, Pharmacodynamic, and Anti-Tumor Activity of SL-172154 As Monotherapy and in Combination with Azacitidine (AZA) in Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) and Higher-Risk Myelodysplastic Syndromes/Neoplasms (HR-MDS) Patients (pts)

Background: CD47 blocking antibodies and SIRPα-Fc fusion proteins have demonstrated activity in combination with azacitidine (AZA) in previously untreated (UnTx) HR-MDS and AML pts. SL-172154 (SIRPα-Fc-CD40L), a hexameric, bi-functional fusion protein consisting of SIRPα domains linked to CD40L domains through an inert Fc linker demonstrated improved anti-tumor activity in comparison to naked CD47 blocking antibodies in pre-clinical studies [de Silva et al. Cancer Immunol Res 2020]. SL-172154, as a CD47 inhibitor, requires combination with AZA to enhance pro-phagocytic signals on leukemic stem cells/blasts thereby potentiating macrophage phagocytosis in AML/HR-MDS. Here we report the results from the Phase 1 Dose Escalation. cohorts for SL-172154 monotherapy [SL-mono] and for SL-172154 + AZA combination [SL-AZA] in pts with HR-MDS or AML. Methods: The objectives of the dose escalation cohorts include evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, pharmacokinetic and pharmacodynamic effects and efficacy per IWG 2006 for MDS and ELN 2017 for AML. SL-172154 was administered IV weekly of a 28-day cycle as monotherapy or with AZA 75 mg/m 2 for 7 days per cycle. mTPI-2 was used for dose escalation with a DLT threshold of 20%. Pts ≥ 18 years old with R/R HR-MDS or R/R AML were eligible for dose escalation cohorts. In addition, UnTx pts with TP53 mutant (TP53m)-MDS were eligible for SL-AZA cohorts. Results: As of May 25, 2023, 37 pts were enrolled (19 in SL-mono; 18 in SL-AZA cohorts): 14 women; median age 70 years [range 44-81]. 27 pts had R/R AML (16 de novo; 11 secondary), 5 had R/R MDS, and 5 were UnTx TP53m-MDS. 23 (85%) of 27 R/R AML pts had adverse genetic abnormalities per 2017 ELN classification, including 9 (33%) with TP53m or del 17. The median number of prior lines of AML therapy was 2 [range 1-4]; 25 (93%) had received venetoclax (VEN) and 23 (85%) had received a hypomethylating agent (HMA). All 5 pts with R/R MDS had previously received HMA and 2 pts were previously treated with VEN. SL-172154 was dose escalated as monotherapy or with AZA as shown in Table 1. Infusion-related reactions (IRRs) were the most common SL-172154-related treatment-emergent AEs (TEAEs) reported in 13 (68%) and 8 (44%) pts in SL-mono and SL-AZA cohorts, respectively. IRRs (51 events) were Grade (G) 1 or 2 except for 3 G3 events (1 each at 6 mg/kg SL-mono and SL-AZA; 1 at 3 mg/kg SL-mono). Other SL-172154-related TEAEs observed in ≥ 3 pts were AST increased (4; 21%), ALT increased (3; 16%) and nausea (3; 17%) in SL-mono cohorts, and nausea (3; 17%) in SL-AZA cohorts. All events of AST/ALT increased were transient. Only one DLT (G3 IRR in the 6 mg/kg SL-AZA cohort) was reported, which required a dose reduction to 3 mg/kg. As of July 10, 2023, in SL-mono cohorts, 1 pt with R/R AML post 7+3, FLAG and VEN/AZA achieved Morphologic Leukemia-Free State (blast reduction from 19% to &amp;lt;5%) after 1 cycle of 6 mg/kg SL-172154 and proceeded to allogeneic hematopoietic cell transplant (allo-HCT) after cycle 2. In 12 evaluable pts with R/R AML receiving SL-AZA, although no objective responses (OR) were observed, relative reduction in bone marrow (BM) blasts from baseline was reported in 2/5 pts at 1 mg SL-AZA (-50%, -75%) and 5/7 pts at 3 mg SL-AZA (ranging from -35% to -90%). One pt underwent an allo-HCT. In 4 evaluable pts with UnTx TP53m-MDS, the ORs were 1 complete remission (CR), 1 marrow CR, and 2 stable disease. 2 of them proceeded to allo-HCT. SL-172154 induced elevations in serum IL-12p40, IP-10, IL-8, IL-10, MIP3α and MCP1: greater response at 3 mg/kg compared to 1 mg/kg while similar between 3 and 6 mg/kg. In the BM, SL-172154 induced a dose-dependent increase in phagocytic cells (CD11b, HLA-DR, CD16, CD36 and CD64) within mature myeloid immune cell compartments (CD45highCD34-). Reduction in leukemic blasts (CD45lowCD34+) was associated with an increase in mature myeloid and phagocytic cell phenotypes (3 mg/kg SL-AZA &amp;gt; 1 mg/kg SL-AZA). Conclusions: SL-172154 was well tolerated up to 3 mg/kg as monotherapy and in combination with AZA. Preliminary efficacy signals were detected in UnTx TP53m-MDS and R/R AML. A response to SL-172154 monotherapy, dose-dependent increases in serum cytokines and accumulation of mature myeloid cells in BM suggest a potential role for CD40 stimulation. Based on the safety, preliminary efficacy and pharmacodynamic activity, 3 mg/kg SL-172154+ AZA is being evaluated in treatment naïve pts with TP53m AML and HR-MDS.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Prognostic Impact of Chromosomal Abnormalities in TP53-Mutated Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndromes

Background: Background: Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) exhibit clinical heterogeneity, and genomic profiles play a crucial role in disease characteristics and survival outcomes. Elli Papaemmanuil et al. classified AML into 11 genomic subgroups (NEJM, 2016), where TP53 mutations and aneuploidy predicted dismal survival. Cases with TP53 mutations are often accompanied by complex chromosomal abnormalities (CAs), but the prognostic impact and target genes of CAs have not been fully studied. Methods: To clarify this issue, specimens of AML and MDS with excess blasts (MDS-EB) were collected from 1,001 patients enrolled at collaborating institutes of Kyoto Hematology Study Group between July 2017 and January 2023. Targeted capture sequencing with 1,216 single nucleotide polymorphism sites was used to analyze primary samples, allowing assessment of copy number alteration (CNA) profiles. The target gene panel included common mutations in myeloid neoplasms. Mutation calling was performed using the established pipeline Genomon 2. Prognostic impact of CNA, single nucleotide variants, and insertion/deletion with 10% or more frequency was evaluated, and the frequencies of CNA were calculated for the short and long arms of chromosomes 1-22 and the X chromosome. Univariate and multivariate analyses were conducted using statistical significance criteria of p &amp;lt; 0.05. Results: The average sequencing depth was 548. Among 1,001 patients, 224 (22.4%) harbored TP53 mutations. TP53-mutated cases had a median age of 70 (24-95) years old at diagnosis, with 117 (52.2%) being AML and 107 (47.8%) MDS-EB. TP53 biallelic mutation, defined by 2 or more mutations in TP53 or loss of heterogeneity of TP53 locus, was present in 200 (89.3%) of cases, more commonly associated with 3 or more CAs or CNA (complex karyotype (CK)-like) than monoallelic mutation (99% vs. 42%, p = 1.9x10 -13). The most frequent affected lesions were del(5q), del(7q), del(7p), del(12p), del(18q), gain(11q), and gain(21q) with frequencies of 76%, 59%, 34%, 33%, 28%, 27%, and 26%, respectively. In survival analysis, gain(21q), gain(19p), del(16q), del(3q), del(19p), del(13q), and del(4q), along with diagnosis of AML ( p = 2.0x10 -2), TP53 biallelic mutation ( p = 6.8x10 -3), and CK-like ( p = 3.4x10 -3), were associated with poor outcomes in univariate analysis. Multivariate analysis revealed gain(19p), del(3q), del(13q), del(4q), diagnosis of AML, and CK-like as independent poor prognostic factors. Here we have defined gain(19p), del(3q), del(13q), and del(4q) as “Risk CNA”. A significant difference in outcomes was observed between patients with and without Risk CNA (n = 110 and n = 114, respectively, Hazard ratio = 2.6, p = 6.0x10 -8): The 6-month survival rate was 70 (95% Confidence interval [60-79]) % for patients without Risk CNA and 42 [31-52] % for those with Risk CNA. Patients with two or more Risk CNAs had worse prognosis than those with one ( p = 2.7x10 -2), indicating independent or additive contributions to outcomes. Similar trends were observed when limiting the analysis to TP53-biallelic cases. Further investigation of Risk CNA target genes was performed by identifying commonly amplified/deleted lesions. Target genes of gain(19p) included DNM2, CARM1, SMARCA4, LDLR, and EPOR, while RB1 and TET2 were potential candidates of del(13q) and del(4q), respectively. Identification of candidate genes for del(3q) was challenging due to broad common deleted lesions. Conclusion: Chromosomal abnormalities stratify the prognosis of AML and MDS-EB with TP53 mutation. The number of Risk CNA also impacts survival. Investigation of target genes of Risk CNA suggests potential therapeutic targets for TP53-mutated myeloid neoplasms, warranting further study for treatment optimization.

Selective Targeting of TP53-Y220C Mutant AML By PC14586 Results in TP53 Wild-Type Conformation and Synergistical Apoptosis Induction By Concomitant Inhibition of Xpo-1, MDM2, or Bcl-2

Patients with TP53-mutant acute myeloid leukemia (AML) constitute the worst outcome group with a survival of only several months. TP53 mutations frequently become dominant following exposure to chemotherapeutic agents, Bcl-2 or MDM2 inhibitors, and confer resistance to multiple other therapies. Y220C is a recurrent hotspot TP53 mutation observed in solid tumors and hematological malignancies, predominantly in AML and MDS (Gener-Ricoset al., ASH 2022). PC14586 (PMV Pharmaceuticals) is designed to bind to the p53-Y220C mutant and restore wild-type (WT) p53 protein conformation and function. PC14586 has been shown to convert Y220C-p53 mutant into WT-p53 protein conformation, induce the activation of p53 transcriptional pathways resulting in selective inhibition of proliferation ofTP53-Y220C mutant solid tumor cell lines and achieved partial responses and stable disease in an ongoing phase I clinical trial in solid tumors (Dumbrava, ASCO 2022, NCT04585750). However, the activity of PC14586 in TP53-Y220C mutated AML/MDS has not been investigated, either as monotherapy or in mechanism-based drug combinations. We investigated the mechanisms of action of PC14586 in AML using cells with TP53-WT, knockout (KO), and mutations (one allele with Y220C or R175H and the other truncated) generated by CRISPR (Boettcher et al., Science 2019) and found that PC14586 converts Y220C mutant p53 to the active form of p53 and selectively activates p53 signaling proteins including MDM2 and p21 in TP53-Y220C AML cells. PC14586 decreased cell viability but had limited apoptogenic activities in the TP53-Y220C AML cells . In primary AML cells with Y220C mutations, PC14586 induced cell death in bulk AML cells and, importantly, also in stem/progenitor cells in 2/3 samples. We reported previously that sustained nuclear retention of p53 by XPO-1 inhibition greatly enhances the transcriptional activity of activated p53 and that activation of p53 by inhibiting its negative regulator MDM2 effectively induces cell death in TP53-WT AML (Kojima et al., Blood 2013, 2005), and that XPO-1 inhibition vastly increased apoptosis induced by MDM2 inhibition through 20-60-fold increased p53 transcriptional activities. We also reported that combined Bcl-2 inhibition and p53 activation is synthetically lethal in TP53-WT AML (Pan et al., Cancer Cell 2017). We hypothesized that XPO-1 synergizes with PC14586 by retaining PC14586-activated p53 in the nucleus and that inhibition of MDM2 induced by PC14586-mediated p53 activation or inhibition of Bcl-2 synergizes with PC14586 in TP53-Y220C AML cells. Indeed, we found that PC14586-induced p53 target proteins were further upregulated when PC14586 was combined with XPO-1 or MDM2 inhibition. Inhibition of XPO-1, MDM2, or Bcl-2 synergistically induced massive apoptosis and decreased cell viability in PC14586 treated TP53 Y220C mutant AML cells (Fig.1), in primary AML blasts, and in CD34+CD38- stem/progenitor cells but not in normal bone marrow cells or stem/progenitor cells through conformational changes of mutant p53 to p53-WT. While PC14586 had limited activity on proliferation in TP53-Y220C AML cells, the combination with Bcl-2 inhibition largely blocked G1-S transition and enhanced apoptosis. The G1-S transition block and apoptosis induction were even more pronounced when PC14586 was combined with XPO-1 or MDM2 inhibitors as determined by flow cytometry measurement of DNA content, and PARP cleavage. Experiments in TP53-Y220C AML PDX models are ongoing and will be reported. In summary, PC14586 converts Y220C-mutant p53 into WT-p53 conformation and induces p53 target proteins. Mechanism-based combinations with XPO-1, MDM2, and Bcl-2 inhibitors induce massive apoptosis. PC14586 is presently in early clinical trials.

Characteristics and Clinical Outcomes of Patients with Acute Myeloid Leukemia with TP53 Mutations - a Single Center Experience

Introduction TP53-mutated acute myeloid leukemia (AML) carries a dismal prognosis. Optimal induction therapy is uncertain, and it remains unclear whether allogeneic stem cell transplant (allo-SCT) improves survival. Methods We investigated 89 cases of TP53-mutated AML diagnosed at or referred to Thomas Jefferson Hospital between April 2016 and May 2023 and reviewed clinical characteristics and outcomes. Remission status was determined by bone marrow biopsy and aspiration after at least 1 cycle of anti-leukemic therapy (ALT). ALT was classified into high- or low-intensity treatment. High-intensity treatment was defined as regimens containing high-dose cytarabine (≥ 200 mg/m 2/day) for at least five consecutive days in combination with an anthracycline. Low-intensity treatment was defined as hypomethylating agent (HMA)-based therapy containing either azacitidine or decitabine with or without venetoclax. Results Median age at diagnosis was 66 (range, 23-91). Forty-nine percent had de novo AML, and the rest had secondary AML. Ninety-one percent had newly diagnosed AML and 9% were found to have a TP53 mutation at relapse. Median somatic mutation count as detected by next-generation sequencing (NGS) was 2 (range, 1-9). Thirty percent were treated with high-intensity treatment, 46% with low-intensity treatment, and 22% received supportive care only. Detailed patient characteristics are in Table 1. Median overall survival (OS) for the entire group was 3.17 months [95% confidence interval (CI) 1.90-5.13 months], and 5.13 months (95% CI 3.83-8.00 months) for patients who received any ALT. Of 69 patients who received ALT, composite complete remission (CRc) rates were 26% with high-intensity treatment and 34% with low-intensity treatment ( p = 0.56). Fifty-six percent did not achieve a remission, and 12% were not able to be assessed due to early mortality. The 30-day mortality was 15% and 7% with high- and low-intensity treatments, respectively. OS was not different between patients who received high- and low-intensity treatments (5.13 months vs 5.27 months, p = 0.65). Twenty-four percent underwent allo-SCT with a median follow-up of 15.2 months. The median time to transplant was 180 days from time of diagnosis (range, 119-341). Median hematopoietic cell transplantation-specific comorbidity index was 3 (range, 1-7). The majority of patients (69%) received a reduced intensity or non-myeloablative regimen, and 63% received a haploidentical transplant. Eighty-one percent of transplant recipients were in CR or CR with incomplete count recovery at the time of transplant; the remaining had either primary induction failure or relapsed disease. Median OS was significantly longer for patients who received allo-SCT compared to those who received chemotherapy only (18.0 months vs 3.7 months, p &amp;lt;0.0001) (Figure 1). Cumulative incidence of relapse post-transplant was 36% at 1 year and 79% at 2 years. Conclusions TP53-mutated AML is associated with a dismal outcome. In our single-center analysis, approximately one-third of patients achieved remission after initial ALT. Consolidation with allo-SCT is associated with improved survival, however relapse risk remains high. Treatment strategies aimed at reducing disease relapse post-transplant are warranted.

Single-Cell Genomics and Proteomics Reveals Venetoclax-Resistant Monocytic Differentiation of TP53 LOH Clones in TP53 Mutant AML

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of abnormal hematopoietic stem and progenitor cells. TP53 mutations, present in 8 - 30 % of AML, are associated with high risk of relapse and poor prognosis. The mechanisms underlying their persistence are not well understood. Here, we attempt to describe the clonal architecture and immunophenotypes of TP53 mutant ( TP53-mut) clones during therapy. Leveraging advancements in single-cell sequencing and machine learning models, this study integrated single-cell mass cytometry (CyTOF) and single-cell DNA + protein sequencing (seq) profiling of ten paired pre- and post-treatment samples from five TP53-mut AML patients. Methods: Patients 1 and 3 received treatment with CD47AB (Magrolimab), 5'-Azacitidine (Aza), and Venetoclax (Ven); Patient 2 received IMGN632 (CD123-targeting ADC), Aza and Ven. Patient 4 received p97 Inhibitor CB-5339; Patient 5 received CD47 inhibitor (ALX148), Aza, Ven. Consequently, Ven/Aza served as the therapeutic backbone for most patients. For single cell DNA+protein analysis, we sequenced a total of 44,550 cells from these 10 samples. Samples with at least 70% viability were stained with the Total-Seq™-D Human Heme Oncology Cocktail, containing 45 oligo-conjugated antibodies for surface markers relevant to AML. For mutation analysis, we utilized a validated AML custom panel (Morita et al. 2020) covering recurrent mutations in 37 genes. For CyTOF, we multiplexed samples and stained with 50 antibodies characterizing lineage, apoptosis, and signaling pathways. We successfully constructed a machine learning model for integrating both data types using our gradient boosting algorithm established pipelines and were able to predict DNA mutation status in CyTOF data with over 95% accuracy. Results: Analysis of the clonal architecture revealed the presence of TP53 mutations not only in AML blasts but also in lymphocyte subpopulations in all TP53-mut clones from the patients in this study. For example, in patient 1, as a representative case, TP53 mutations were observed in 16.5% of CD4+ T cells, 35.9% of CD8+ T cells, and 19.2% of NK cells. Clonal evolution analysis showed sensitivity of TP53-mut clones with TP53 Loss of heterozygosity ( TP53 mut + LOH) to Ven/Aza and monocytic differentiation in resistant cells, potentially a manifestation of their stem-like nature. Patient 1 displayed three distinct subclones: clone 1 harbored an ETV6 mutation (p.V322S), clone 2, derived from clone 1, acquired additional IDH1 (p.R132L) and TP53 (p.R241G) mutations, clone 3, originating from clone 2 and comprising the highest proportion, exhibited loss of the other TP53 allele (LOH), and decreased from 96.2% to 55.2% post-treatment (Figure 1A). Meanwhile, clone 1 and clone 2 increased from 1.9% to 27.3% and 1.9% to 17.4% respectively. Dissecting the phenotypic changes, clone 3 showed significant pre- and post-treatment shifts in cellular composition. Pre-treatment, it consisted predominantly of 89.7% CD34+ blasts and only 0.2% CD16+ monocytic cells. Post-treatment, CD34+ blasts decreased to 9.3%, while CD16+ monocytic cells increased to 67.8%. CyTOF analysis corroborated our findings (Figure 1B) with trajectory analysis revealing that AML cells had a stemness profile (CD34+CD38- and C-kit+) located at the beginning of the trajectory. Conversely, post-treatment AML cells displayed a loss of stemness and expressed myeloid differentiation markers, including CD33, CD11b, CD14, and CD68. We concluded that Ven-based treatments induce monocytic differentiation in resistant AML cells. CyTOF analysis of the proteomic profiles of resistant cells revealed an increase in Ki67, BCL-XL, and 4EBP1 and a decrease in CD34 and C-KIT, suggesting a potential resistance mechanism involving the upregulation of anti-apoptotic molecules and mTOR pathway. Notably, CD36, a fatty acid transporter, exhibited increased expression in monocytic cells, suggesting an altered metabolism in the survival monocytic cells. Conclusion: This study establishes a genotype-phenotype connection through single-cell proteogenomic profiling of TP53-mutated AML, describing the clonal evolution and immunophenotypic dynamics during treatment while proposing a potential mechanism of resistance.

Myeloid Neoplasia in Patients with Hereditary Hemoglobinopathies and Hemolytic Anemias (HHAs)

Background: Studies have shown an increased incidence of myeloid neoplasia (MN) in patients with sickle cell disease (SCD). It is hypothesized that increased erythropoietic stress may influence acquisition of molecular drivers of MN in SCD. However, present understanding of MN presentation and outcomes in SCD and other hereditary hemoglobinopathies and hemolytic anemias (HHAs) is limited to case reports. Systematic evaluation of MNs in patients with HHAs will better contextualize the risk of MN in this population relative to non-HHA controls. Methods: Patients with HHAs were identified from the Mass General Brigham Research Patient Data Repository using ICD-10 codes. Evaluated HHAs included sickle cell disease (SCD), sickle cell trait (SCT), thalassemia major, thalassemia trait, other hemoglobinopathy traits, and hereditary spherocytosis or elliptocytosis. A final HHA-MN cohort included patients with laboratory confirmed HHA diagnoses as well as incident myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), Ph+ chronic myelogenous leukemia (Ph+ CML), Ph negative myeloproliferative neoplasms (Ph- MPNs) or acute myeloid leukemia (AML). A control cohort of non-HHA MN patients was obtained from the Dana-Farber Hematologic Malignancies Data Repository. Clinical, molecular, and survival data were summarized and compared between HHA-MN and controls. Results: The HHA-MN cohort included 62 patients with HHAs and incident MN (Table 1). HHAs included SCD (n = 6, 9.7%), SCT (n = 9, 14.5%), thalassemia trait (n = 36, 58.1%), thalassemia major (n = 2, 3.2%), hereditary spherocytosis or elliptocytosis (n = 4, 6.5%), and other hemoglobinopathy traits (n = 5, 8.1%). MN was diagnosed at a median age of 61 years [IQR: 48, 70] in the HHA-MN cohort and incident MNs included AML (n = 12, 19.4%), MDS/CMML (n = 22, 35.4%), Ph+ CML (n = 6, 9.7%), and Ph- MPNs (n = 22, 35.4%). A control population of 3388 MN patients without HHA included cases of AML (n = 754, 22.2%), MDS/CMML (n= 1272, 37.5%), Ph+ CML (n = 354, 10.4%), and Ph- MPNs (n = 1008, 29.7%). Compared to HHA patients, control patients were older at MN diagnosis (median age 65 years [IQR: 56, 73], p = 0.0071), driven by significantly younger ages at AML diagnosis for HHA-MN patients (median age 50 years [IQR: 33, 63] vs 65 years [IQR: 57, 73], p =0.0044). AML was the most common MN in SCD (n = 4, 66.7%) whereas incident MDS/CMML was the predominant MN among individuals with thalassemia trait (n = 12, 33.3%) or hereditary spherocytosis and elliptocytosis (n = 2, 50%). Diagnostic cytogenetic data were available for 45 patients in the HHA-MN cohort (72.6% of cohort). Complex karyotype, defined as 3 or more chromosomal aberrations, was observed in 3 of 11 (27.3%) AML, 3 of 19 (15.8%) MDS/CMML and 1 of 17 (5.9%) Ph- MPN cases. Of the 33 HHA-MN patients with available diagnostic next generation sequencing, 60.6% had 2 or more pathogenic mutations at MN diagnosis. Including 2 cases of TP53-mutant AML in patients with SCD, the majority (90%) of AML and 38.9% of MDS/CMML diagnosed in HHA-MN patients were intermediate to adverse risk by ELN criteria or intermediate to very high risk by IPSS-R, respectively. High risk disease was also common in our control MN cohort, where 88.3% of AML cases were intermediate or adverse risk by ELN criteria and 55.9% of MDS/CMML cases were intermediate to very high risk by IPSS-R. Survival varied by subtype of HHA and MN (Figure 1). Survival was lowest for SCD (33.3% at 60-months) and highest for thalassemia traits (67.1% at 60 months). In HHA patients compared to controls, 60-month survival was 65.0% overall (vs 56.4%), 14.3% for AML (vs 23.8%), 51.0% for MDS/CMML (vs 43.9%), 90.5% in Ph- MPN (vs 80.6%), and 100% in Ph+ CML (vs 89.4%). Within the limitations of our sample size, survival differences were not statistically significant. Conclusion: In this retrospective analysis, we observe younger age of diagnosis of myeloid malignancy for patients with HHAs compared to non-HHA controls. Inferior survival in SCD and thalassemia major is largely explained by a higher proportion of AML diagnoses in patients with SCD. AML and MDS/CMML diagnosed in HHA patients tended to be intermediate or high-risk disease, with survival similar to the non-HHA control population that was also enriched for higher risk patients. Validation of our findings in an expanded cohort of HHA patients is currently underway.