TP53-mutated Acute Myeloid Leukemia Research Articles

Novel investigational approaches for high-risk genetic subsets of AML: TP53, KMT2A, FLT3.

The treatment landscape in acute myeloid leukemia (AML) is rapidly evolving, with multiple new therapies approved in recent years. However, the prognosis for patients with high-risk genetic subsets of AML remains poor, and the development of more effective treatment options for these patients is ongoing. Three of these high-risk AML patient subsets include TP53-mutated AML, FLT3-internal tandem duplication (ITD)-mutated AML, and AML harboring rearrangements affecting the KMT2A locus (KMT2A-r AML). The prognosis for TP53-mutated AML remains poor with both intensive and targeted regimens, including those incorporating the BCL-2 inhibitor, venetoclax. Allogeneic hematopoietic stem cell transplantation is the only potentially curative therapy for these patients, but posttransplant relapse rates remain high. Patients with FLT3-ITD-mutated AML continue to have suboptimal outcomes with standard therapies and experience high rates of relapse following transplant. KMT2A-r AML is also associated with poor outcomes with current treatment approaches, and effective standards of care are lacking for patients with relapsed/refractory disease. This article discusses current treatment approaches, along with the investigational agents being explored for the treatment of these 3 AML subsets, focusing primarily on agents that are further along in development.

Targeting the Mevalonate Pathway in the Mitochondria-Dependent Chemotherapy Resistance of TP53 Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) bearing mutations in the TP53 tumor suppressor gene is the most fatal of AMLs with a two-year overall survival between 0-15%. Patients with TP53 mutant (MT) AML typically respond to initial therapy but quickly relapse and die of disease due to chemotherapy resistance. We have undertaken to understand the mechanisms of chemotherapy resistance in TP53 MT AML. Initially, we purified AML blasts to 99% purity using flow sorting and performed RNA sequencing on TP53 MT and wildtype samples. Gene set enrichment analysis demonstrated that TP53 MT AML patient samples were enriched in the cholesterol synthesis or mevalonate pathway. This was validated using the BEAT AML dataset. Interestingly, p53 loss and/or mutation in breast, liver, colon and pancreatic cancer models leads to a dependency on an upregulated mevalonate pathway. Despite these data, the role of the mevalonate pathway in TP53 MT AML is unknown. We have subsequently explored the role of the mevalonate pathway in TP53 MT AML chemotherapy resistance. Experiments were performed in CRISPR-engineered TP53 mutated human AML cells with isogenic controls. Treatment with DNA damaging agents and analysis for cell survival confirmed that loss of wildtype p53 function enhanced cell survival. Recent results have demonstrated that chemotherapy resistance in AML cells is associated with an increase in oxidative phosphorylation (OXPHOS), which is dynamically induced by DNA damage. We confirmed that TP53 MT AML cells increase OXPHOS after DNA damage (Figure 1). Treatment of cells with rosuvastatin, a potent inhibitor of the mevalonate pathway, blocked this increase in OXPHOS and induced chemosensitivity. Lipidomic analysis demonstrates that the increase in OXPHOS correlates with an increase in coenzyme Q10 (CoQ10), a byproduct of the mevalonate pathway. Importantly, co-treatment of cells with a soluble form of mevalonate or a downstream byproduct, geranylgeranyl pyrophosphate (GGPP), recovered OXPHOS and subsequent chemoresistance. This indicates rosuvastatin's activity is specific to the mevalonate pathway, independent of cholesterol, and dependent on the GGPP branch of the pathway. Analysis is ongoing to determine the role of GGPP in regulating the mitochondrial response to DNA damage in TP53 MT cells, including if GGPP is required for CoQ10 synthesis as this remains a controversy in the literature. 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 2). To determine if this model is physiologically relevant, we studied a patient derived xenograft (PDX) model of TP53 MT AML cells. Engrafted mice were treated with standard AML chemotherapeutic agent, cytarabine (AraC), for five days alone or in combination with high dose rosuvastatin. Interestingly, mice sacrificed three days after completing AraC administration continue to demonstrate an enhanced OXPHOS response. This response is blunted by co-treatment with rosuvastatin. As expected, TP53 MT AML cells respond modestly to AraC alone and this response was increased with the combination therapy. Overall, these results demonstrate that TP53 MT AML requires the mevalonate pathway for chemotherapy resistance and targeting of the mevalonate pathway in combination with chemotherapy may improve clinical responses. Through detailed biochemical analysis of this mechanism, we will elucidate novel therapeutic approaches to TP53 MT AML. Figure Legend: (1) Oxygen consumption rate measured by seahorse technology in indicated cell lines and conditions. Statistics performed with unpaired T-test in GraphPad with stars indicating significance as per figure. (2) Proposed schema of mevalonate pathway dependencies in TP53 mutant (MT) AML. Bold arrows indicate increased byproduct dependency in TP53 MT AML cells treated with DNA damaging chemotherapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Discrimination within a Heterogeneous Population of TP53-Aberrant Myelodysplastic Syndromes and Acute Myeloid Leukemia

Background:TP53-aberrant myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) constitute the highest risk molecular subgroup of myeloid neoplasms, and there are currently no FDA-approved targeted therapies for this subset of patients. We have recently explored the reasons for these distinctly adverse outcomes using clonal dynamic modeling. However, prognostic discrimination within this heterogeneous group of patients has not been validated in large-scale efforts. More recently, efforts to gain clarity on this disease subset include the IPSS-M model for MDS, the European LeukemiaNet (ELN) 2022 classification for AML, and the 2022 International Consensus Classification (ICC) for both MDS and AML. For example, the IPSS-M model has shown an adjusted hazard ratio of 3.27 (95% CI 2.38 - 4.48) for multi-hit TP53 aberration; the ELN 2022 classification now includes a new category termed "AML with mutated TP53" for patients with TP53 variant allele frequency (VAF) ≥ 10%; and the 2022 ICC has designated categories for MDS and AML with TP53 mutation. However, these systems do not discern among the subtypes of TP53 genomic aberrations with regard to clinical outcomes. Methods: Identification of patients from the UMass Chan Leukemia Registry was performed via UMass Clarity, a Microsoft SQL Server database comprised of 23,000 tables, in conjunction with the main reporting environment in EPIC. The Registry identified patients harboring one or more aberrations in TP53 and with ICD-10 code of either D46.9 (MDS, including its subentities) or C92.00 (AML, including its subentities) between 2011-2022. A total of 63 patients were identified. Patients with TP53-aberrant MDS or AML were stratified by the presence or absence of multi-hit status and/or TP53 VAF >50%. Multi-hit status was defined as per the 2022 ICC: two or more distinct TP53 mutations with VAF ≥ 10%, or a single TP53 mutation plus one of the following abnormalities: (1) del(17p13.1), (2) TP53 VAF >50%, (3) copy-neutral loss-of-heterozygosity, or (4) any complex karyotype. Monoallelic status was defined as non-multi-hit. Kaplan-Meier survival with log rank p value analysis was performed on subgroups based on the TP53 genomic state. Results: Median age of the UMass cohort (n = 63) was 69.8 ± 11.3 years. There were 35 patients (55.6%) with AML. We identified 58 patients who met the 2022 ICC definition for myeloid neoplasm with mutated TP53 (after exclusion of 5 patients with VAF < 10%) then stratified patients based on hit status and VAF. Median overall survival (OS) was similar among patients with monoallelic TP53-mutant MDS, multi-hit TP53-mutant MDS, and monoallelic TP53-mutant AML (median OS 440 days vs. 437 days vs. 440 days, respectively) (Panel A). In contrast, patients with multi-hit TP53-mutant AML had distinctly shorter survival (median 46 days) (Panel A). For patients with multi-hit status MDS or AML per the 2022 ICC (n = 41), we sub-stratified patients based on TP53 VAF. OS for patients with MDS with VAF ≤ 50% and VAF > 50% was similar (median 376 days vs. 435 days, respectively) (Panel B). Patients with AML with TP53 VAF > 50% had significantly inferior OS (median 43 days) (Panel B). Twelve (19.0%) of 63 patients underwent allogeneic hematopoietic cell transplant (allo-HCT) (2 had VAF >50%). Allo-HCT significantly improved median OS (732 days with allo-HCT vs. 161 days for n = 51 without allo-HCT; p = 0.0033) (data not shown). Only 3 (25%) of the 12 allo-HCT recipients achieved MRD-free remission lasting beyond 1000 days; VAF was ≤ 10% in all of these long-term survivors with TP53-mutant MDS or AML. Conclusion:TP53-aberrant MDS and AML comprise a prognostically heterogeneous group of patients with inferior clinical outcomes, though the highest risk subgroup appears to be AML with multi-hit TP53 status or AML with TP53 VAF > 50%. There is high value in determining the allelic state and hit status at the time of diagnosis to help prognosticate and develop a long-term management plan. Allo-HCT improves median OS, though durable MRD-negative remission is rare. Our data suggest that there may be a limited window of VAF ≤ 10% for the success of allo-HCT for TP53-aberrant myeloid neoplasm. We acknowledge that there is a selection bias for proceeding with allo-HCT. Large-scale systematic studies are needed to comprehensively profile patients classified as myeloid neoplasm with mutated TP53 such that risk-adapted management strategies can implemented. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Polyploidy Is Necessary for Apoptotic Cell Death in TP53-Mut AML in Response to Polo-like-Kinase 4 (PLK4) Inhibition and Results in Caspase 3 Cleavage

Background Approximately twenty percent of patients with newly diagnosed acute myeloid leukemia (AML) have TP53 mutations, associated with complex karyotype and the highest risk of relapse(Döhner et al., 2017; Giacomelli et al., 2018). Although combination therapies, such as Ara-C + anthracyclines and Venetoclax (VEN) + hypomethylating agents (HMA) result in higher remission rates in patients with TP53-mutant AML, the mutant clone is never eliminated and invariably leads to relapse(Short et al., 2021; Takahashi et al., 2016). Novel targets and mechanisms associated with TP53-mut AML need to be identified. Results: We performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations of TP53-mutant or TP53-wild-type (wt) high-risk AML patients before and after treatment. Amongst overexpressed genes, we identified Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis and therefore of polyploidy, as a potential target highly expressed in TP53-mutant AML using differential gene expression analysis of TP53-wt vs TP53-mutant samples collected (TP53-mutant=36, TP53-wt=31, PLK4 LOG2FC=~2, p<0.001). We validated this finding using an independent RNA-seq dataset from the Munich Leukemia Lab of 726 AML samples (TP53-mutant=72, TP53-wt=654, PLK4 LOG2FC=0.48, p<0.001) and investigated the impact of PLK4 expression levels on AML patient outcomes in the context of TP53-status. High levels of PLK4 expression in AML are associated with poor outcomes (Figure 1). Targeting PLK4 by CFI-400945 (25nM, Treadwell Therapeutics, Toronto, Canada) in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 for 72 hours resulted in increased polyploid cells in TP53-mut cells compared to TP53-wt AML (> 2 fold, p< 0.0001). PLK4 inhibition triggered higher levels of cleaved caspase 3 in polyploid TP53-mut polyploid MOLM-13 cells compared to TP53-wt MOLM-13 cells (Figure 2). Polyploidization in TP53-mut MOLM-13 cells 48 hours post treatment with 15nM of PLK4 inhibitor was not reversible and retained higher apoptogenic activity in TP53-mut MOLM-13 cells even after wash out for additional 48 hours. To translate our findings to in vivo models, we transplanted CRISPR-generated TP53-wt, TP53-ko, and TP53-248 mutant MOLM-13 cells (Boettcher et al., 2019) into NSG mice (n=58: untreated n= 28, treated n=30), confirmed successful engraftment, and treated the mice with CFI-400945 (oral gavage 7.5 mg/kg, 5 times/week) for 21 days. Our preliminary data indicates that all treated mice showed improved survival compared to untreated mice. The median survival for treated TP53-wt vs untreated TP53-wt was 17.5 vs 14.5 days (20.7% increase, n=20 (untreated n=10, treated n=10), p=0.00086). The median for treated TP53-R248Q vs untreated TP53-R248Q was 15.0 vs 12.0 days respectively (25% increase, n=19 (untreated n=9, treated n=10), p=0.00039). The median OS for treated TP53-ko vs untreated TP53-ko was 11 vs 8 days respectively (37.5% increase, n=19 (untreated n=9, treated n=10), p<0.0001). Next, we performed a Cox proportional hazard model to investigate the contribution of TP53 genotype and PLK4 inhibition on overall survival. The resulting model (Likelihood ratio test=83.09 on 3 degrees of freedom and p<2.2e-16) showed a higher hazard ratio in recipients with TP53-R248Q and TP53-ko (~4.3, and ~1.2 hazard coefficients respectively) in comparison to TP53-wt (p=2.67e-14, and 0.000708 respectively). Additionally, treated mice had statistically significant lower hazard ratios compared to untreated (hazard coefficients=-2.47277 and p=8.38e-10). The data suggest that PLK4 inhibition is a promising treatment approach for AML with TP53 mutation. We are presently testing different dosing schedules in other aggressive TP53-mut AML models to further ascertain the efficacy of PLK4 inhibition. Conclusion: We established an association between PLK4 overexpression and poor survival outcomes in patients with newly diagnosed AML. TP53-mutant AML express higher levels of PLK4 in comparison to TP53-wt AML and targeting PLK4 induces polyploidy and apoptotic cell death predominantly in TP53-mutant AML. We demonstrate that targeting of PLK4 improves overall survival in TP53-mut and TP53-ko AML cells in vivo. Data suggest that targeting PLK4 represents a potential treatment approach for TP53-mut AML. An early phase, multi-center clinical trial is ongoing with CFI-400945 in AML (NCT04730258, TWT-202). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The XPO7/Npat Axis Is a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate below 30%. While AML is a genetically heterogeneous disease, TP53 mutations are among the most powerful risk factors in AML, which underscores the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/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. To generate AML lines with a relatively "clean" genetic background, we established mouse AML lines harboring WT Trp53 with normal karyotype (Yamauchi et al. Cancer Cell, 2018). We then generated Trp53-KO AML lines using a single guide RNA (sgRNA) targeting Trp53. Genome-wide CRISPR-Cas9 dropout screens were performed using these lines to identify genes/pathways whose loss is vulnerable to TP53 deficiency. We identified Xpo7, a putative nuclear/cytoplasmic transporter, as a factor necessary for the survival of Trp53-KO AML cells. sgRNAs targeting Xpo7 were enriched in Trp53-WT AML cells after the 16-day culture, while they were significantly depleted in Trp53-KO cells. Trp53-KO cells were vulnerable to Xpo7 depletion, while Xpo7 functioned as a Trp53-dependent tumor suppressor in Trp53-WT AML cells. As expected, CRISPR/Cas9-mediated Xpo7 depletion and shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. We next performed CRISPR-Cas9 saturation mutagenesis scan targeting all Xpo7 exons using Trp53-WT and -KO mouse AML cells. Strikingly, sgRNAs targeting Xpo7 coding regions were significantly enriched only in the Trp53-WT cells, namely in the presence of an MDM2 inhibitor. In stark contrast, the same sgRNAs which target coding exons including the C-terminal importin-β domain were mostly depleted in Trp53-KO AMLs. Since Xpo7 presumably mediates the nuclear import and export of proteins, we hypothesized that Xpo7 promotes retention of Trp53 protein in the nucleus in Trp53-WT AML cells. As expected, Trp53 protein levels in the nucleus and mRNA levels of Trp53-targeting genes were significantly decreased upon Xpo7 depletion. Furthermore, Xpo7 depletion resulted in resistance to Selinexor, a XPO1 inhibitor that reportedly blocks Trp53 protein export from the nucleus. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Next, to elucidate the mechanisms underlying the toxic effects observed in Trp53-KO AMLs upon Xpo7 depletion, we set out to identify proteins whose cytoplasmic/nuclear localization was potentially regulated by Xpo7 in Trp53-KO AML cells. We first performed IP-mass spectrometry using an anti-Xpo7 antibody to identify Xpo7 interacting proteins. Then, we obtained both cytoplasmic and nuclear protein extracts from both Xpo7-WT and -KO AML cells and identified proteins whose localization was significantly altered upon Xpo7 deletion by mass spectrometry. We found that Npat, an activator of histone transcription in the G1/S transition, interacted with Xpo7 and localized more in the cytoplasm upon Xpo7 depletion in Trp53-KO AMLs to a significant extent. Strikingly, Npat protein levels were robustly upregulated following Trp53 depletion, and Npat depletion significantly delayed the progression of the G1/S transition and proliferation in Trp53-KO AML cells. Finally, to explore the functional significance of the XPO7/NPAT axis in human TP53-mutated AML cells, we assessed the correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 and NPAT mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM, 2013). 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 and Iacobucci et al. Nat Genet. 2019). In fact, shRNA-mediated XPO7 or NPAT knockdown significantly suppressed 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. Our data suggest that XPO7/NPAT inactivation is a therapeutic vulnerability for TP53-mutated AML, such as AEL.

TP53 Mutant Acute Myeloid Leukemia: The Immune and Metabolic Perspective

TP53 mutated/deleted acute myeloid leukemia (AML) stands out as one of the poorest prognosis forms of acute leukemia with a median overall survival not reaching one year in most cases, even in selected cases when allogenic stem-cell transplantation is performed. This aggressive behavior relies on intrinsic chemoresistance of blast cells and on high rates of relapse. New insights into the biology of the disease have shown strong linkage between TP53 mutant AML, altered metabolic features and immunoregulation uncovering new scenarios and leading to possibilities beyond current treatment approaches. Furthermore, new targeted therapies acting on misfolded/dysfunctional p53 protein are under current investigation with the aim to improve outcomes. In this review, we sought to offer an insight into TP53 mutant AML current biology and treatment approaches, with a special focus on leukemia-associated immune and metabolic changes.

A Phase I/II Study of Combination of Azacitidine, Venetoclax and Pevonedistat in Patients with Newly-Diagnosed Secondary Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) with Hypomethylating Agent (HMA) Failure

Background: Pevonedistat (PEV) is an inhibitor of NEDD8-activating enzyme (NAE) which is critical for degradation of several cellular proteins essential for tumor growth and survival. The combination of azacitidine (AZA) and venetoclax (VEN) is approved for patients (pts) with newly diagnosed (ND) AML who are unsuitable for intensive chemotherapy, and clinical studies show preliminary efficacy with AZA+VEN in higher risk MDS pts. We evaluate the efficacy and safety of the combination of AZA, VEN, and PEV in ND secondary AML (s-AML) and in MDS after HMA failure. Methods: This phase 1/2 trial enrolled pts with ND s-AML, including therapy-related AML (t-AML), AML from prior MDS or chronic myelomonocytic leukemia (CMML) or myeloproliferative neoplasm (MPN), or AML with dysplasia in ≥50% cells in ≥2 myeloid lineages (MRC). A second arm enrolled pts with MDS or CMML that had progressed on prior HMA therapy. Pts had to have a performance status ≤2, total bilirubin ≤1.5 x upper limit of normal (ULN), ALT/AST ≤2.5 x ULN, and creatinine clearance ≥30 mL/min. Pts in AML arm may have received prior HMA for preceding hematologic malignancy. In both AML and MDS/CMML cohorts, pts received AZA 75 mg/m2 SC/IV on days 1-7 and PEV 20 mg/m2 IV on days 1, 3 and 5 on every 28 day-cycle. In AML cohort, VEN was given on days 1-28 in cycle 1 and days 1-21 on cycle 2+. In MDS/CMML cohort, VEN was given on days 3-14 in cycle 1 and days 1-14 on cycle 2+. In initial phase 1 portion of the study (AML cohort only), VEN at max dose 200 mg and 400 mg were explored. Results: Between 3/2019 and 8/2021, 32 pts with AML (10 t-AML, 22 AML from MDS/CMML/MPN, 4 MRC) and 8 pts with MDS/CMML post-HMA failure (6 with MDS, 2 with CMML) were treated (Table). In the AML cohort, the median age was 74 years, with 14 pts (44%) ≥75 years of age. Cytogenetics was adverse in 21 pts (66%), and adverse risk mutations, including TP53, RUNX1, and ASXL1 were present in 34%, 31%, and 22%, respectively. 17 pts (53%) had treated secondary AML (ts-AML; i.e. prior exposure to HMA or chemotherapy for preceding myeloid neoplasm). The overall response rate (ORR, defined as CR+CRi+MLFS) was 75%, with CR in 15 pts (47%), CRi in 6 pts (19%), and MLFS in 3 pts (9%). One pt achieved PR (3%). 78% of pts achieved best response after 1 cycle of therapy (range, 1-4 cycles to best response). Among 21 pts with CR/CRi, 10 pts (48%) achieved MRD negativity by flow cytometry. 14/21 pts (67%) with poor-risk cytogenetics responded, compared to 10/11 pts (91%) with non-poor cytogenetics. 9/11 pts (82%) with TP53-mutated AML responded. 11/17 pts (65%) with ts-AML responded, compared with 13/15 pts (87%) who did not have ts-AML. With median follow up of 20 months (mos) for the AML cohort, the median OS was 8.2 mos and median relapse-free survival (RFS) was 7.4 mos. 5 pts proceeded to transplant in first remission. The median OS for pts with adverse cytogenetics was 6.8 mos and was not reached in pts without adverse cytogenetics (1-year OS of 70%). The median OS for pts with TP53-mutated AML (n=11) was 8.1 mos, pts with inv(3) (n=4) was 3.8 mos, and pts without TP53 mutation or inv(3) (n=17) was 18 mos (Figure). In MDS/CMML HMA failure cohort, the median age was 73 years. Most pts had adverse cytogenetics (50%) and were high to very high-risk by IPSS-R (87%). Overall response rate (CR+marrow CR [mCR]+HI) was 75%, with CR in 3 pts (37%), mCR in 2 pts (25%), and HI in 1 pts (13%). With a median follow up of 11 mos, the median OS was 9.9 mos. Transformation to AML occurred in 1 pt (after 11 mos). No DLTs were observed in the phase 1 portion, and VEN 400 mg daily was the recommended phase 2 dose. Myelosuppression was similar to expectations with AZA+VEN alone. Grade 3/4 non-hematologic adverse events occurring in ≥10% pts were febrile neutropenia or other infection in 75%, hypophosphatemia in 22%, and hyperbilirubinemia in 10%. The 4-week and 8-week mortality rates were 9% and 25% in AML cohort and 0% and 13% in MDS/CMML cohort, respectively. Conclusion: AZA, VEN, and PEV combination is active and well-tolerated in both ND s-AML and MDS/CMML after HMA failure. This population was enriched with very poor risk features, including a majority of pts in the AML cohort with prior HMA or chemotherapy exposure for an antecedent myeloid malignancy and 1/3 of pts with a TP53 mutation. Response rates were high across disease subgroups, although duration of responses and survival were modest in pts with historically poor risk features, including those with adverse risk cytogenetics and TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Outcomes of Patients with TP53-Mutated AML after First Relapse or with Primary Refractory Disease: Results from Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND)

BackgroundTP53-mutated (m) acute myeloid leukemia (AML) is a high-risk disease with sub-optimal response to conventional therapies and portends a particularly worse outcome in patients with relapsed or refractory disease. In this study we analyzed the outcomes of patients with TP53m AML after first relapse or after primary induction failure. Methods We conducted this retrospective study through COMMAND (Consortium on Myeloid Malignancies and Neoplastic Diseases), a collaboration involving 10 US academic institutions. 366 patients with TP53m AML diagnosed between 2012-2021 were evaluated with 151 patients who had either primary refractory (87 [58%]) or relapsed (64 [42%]) disease and received further care, were the subject of this study. Continuous variables were summarized as median (range), while categorical variables were reported as frequency (percentage). Predictors of treatment response were assessed by Chi-square or Fisher's exact test for nominal data and Wilcoxon rank-sum test for continuous variables. Event free survival (EFS) was estimated from time of diagnosis to progression or death. Overall survival (OS) from the date of first salvage until last follow up or death was evaluated by the Kaplan-Meier method with differences compared by the log-rank test. Cox proportional hazards regression models were used to find the univariate and multivariate predictors of OS. Multivariable models included all significant univariate predictors with p value <0.1. Results Baseline characteristics Baseline characteristics and treatment outcome are summarized in Table 1. The median age of the patients was 64 (range [R], 18-84) years and 59 (39%) patients were > 65 years of age. 21% of patients had therapy-related AML, 86% had complex cytogenetics (CG) and 9% of these patients had erythroid leukemia. Twenty-one (14%) patients relapsed after allogeneic stem cell transplantation (alloHCT). Co-mutations were observed in 45% of patients with the most frequently occurring (≥ 5% of patients) being ASXL1 (5%), TET2 (7%), DNMT3A (8%), RUNX1 (7%), RAS (7%), and JAK2 (8%) mutations. Salvage therapies were venetoclax-based (with hypomethylating agent [HMA] or low dose cytarabine [LDAC]) (46%), HMA +/- investigational agent (20.5%), intensive chemotherapy (IC; excluding CPX-351) (17%), other low intensity +/- investigational agent (11%) and CPX-351 (6%). Responses The rate of complete remission (CR) with or without count recovery (CRi) was 24% (n= 35) and 13% (n=20) were able to receive alloHCT after achieving response. We evaluated predictors of response (CR/CRi) using baseline variables. Age > 65 yrs (23% vs 77%, p=0.03) and secondary AML (15% vs 85%, p= 0.02) were significantly associated with an inferior response rate. Remaining variables were not significantly associated with response to salvage therapy. Survival The median EFS was 3.97 months (95% CI: 31.5-4.78) and median OS was 4.37 months (95% confidence interval [CI]: 3.58-5.15) (Figure 1a). In sub-group analysis, median OS (in months) based on salvage therapy was 3.23, 5.20, 3.93, 3.37 and 2.83 with venetoclax based, HMA +/- investigational agent, IC, other low intensity +/- investigational agent and CPX-351, respectively (p= 0.16) (Figure 1b). We did multivariate analysis (MVA) for OS, utilizing univariate predictors with p value <0.1. Achievement of CR/CRi (hazard ratio [HR]=0.57, 95% CI: 0.36-0.91; p=0.01) and alloHCT after salvage therapy (HR=0.45, 95% CI: 0.24-0.75; p= 0.03) retained significance for better OS. Complex CG (HR=1.83, 95% CI: 1.05-3.17; p= 0.03) and diagnosis of erythroid leukemia (HR=2.25, 95% CI: 1.22-4.17; p= 0.009) retained significance for inferior OS in MVA. Conclusion As expected, our study confirms the dismal outcome of patients with TP53m AML progressing on first line therapy, however alloHCT can significantly improve outcome even after salvage therapy in a subset of patients who achieve response and are eligible for alloHCT. Effective therapies are urgently needed to improve outcome of patients with TP53m AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High MN1 Expression Is Associated with an Lspc-Enriched Phenotype and Glycolysis Representing a New Vulnerability in Acute Myeloid Leukemia

Despite a steady increase in the number of targeted therapies over the past decades, the 5-year overall survival (OS) rate remains poor in acute myeloid leukemia (AML). Currently, high expression of the oncogenic Meningioma 1 (MN1) is associated with poor OS in AMLs with normal karyotype, and we correlated its expression with a more immature cellular composition in AML samples. Hence, we hypothesized that supplementation of AML cells that usually fail to engraft in xenograft models, such as acute promyelocytic leukemia (APL), with MN1 would promote in vivo expansion in mice allowing the study of potential therapeutic vulnerabilities. The in vitro experiments were performed in human AML cell lines, primary AML cells (n=39), and isolated healthy CD34+ cells. MN1-overexpression (OE) was lentivirally performed. Bioenergetic profiles were evaluated by a Seahorse analyzer. Gene expression was measured by RNA-sequencing/qPCR. Protein levels were measured by immunofluorescence microscopy. Viability and surface protein expression was evaluated by flow cytometry. For in vivo studies, eight-weeks old female NSGS mice were transplanted with MN1-OE primary APL blast cells, and mice were followed for 12 weeks. Cells from the mouse organs were isolated, analyzed, and sorted for human APL blasts (CD45+CD117+CD33+) for ex vivo studies. We show that genetic MN1-OE in APL cells resulted in increased cell survival upon cytotoxic therapy, and decreased sensitivity to ATRA-induced terminal differentiation in short and long term cultures (3/8 days). In healthy CD34+ cells, we observed that MN1-OE was able to increase cell proliferation and stemness properties in CMP-sorted (CD34+CD38+CD123+CD45RA-) but not in GMP-sorted cells (CD34+CD38+CD123+CD45RA+), showing in human models the results from Heuser et al., 2011 in murine MN1-model. Co-expression of MN1+PML-RARα in healthy CD34+ cells resulted in superior expansion with retention of the CD34 marker, in comparison with the single hits. At the molecular level, APL cells with MN1high displayed transcriptional programs associated with "HYPOXIA"/"HSC-UP", whereas MN1low levels correlated with "OXPHOS-UP"/"GMP-UP". Despite no evident differences in Seahorse analysis, MN1-OE cells exhibited increased levels of the glycolytic enzyme PDK1. Functionally, MN1-OE cells were more sensitive to glycolytic inhibitors 2,2-dichloroacetophenone (a PDK1 inhibitor) and 2-deoxyglucose, revealing glycolysis as a metabolic vulnerability in MN1high AMLs, which was further validated in primary AML samples. These data are in line with our previous report indicating that more immature AML subtypes typically display a more glycolytic metabolic profile (Erdem et al, 2022). MN1 is an intrinsically disordered protein containing a long polyQ-stretch that is required for transformation (Riedel et al, 2021). We questioned whether the expression of chaperone proteins would be necessary for the appropriate functioning of MN1. We observed that expression of the DNAJB6 isoform 2 (DNAJB6b) correlated with MN1 expression across a large panel of genetic subtypes of AML, including more immature AML subtypes, such as the TP53-mutant AML but also in APL. shRNA-induced knockdown in AML cells of DNAJB6b resulted in cytosolic retention of MN1, associated with reduced cell survival. In vivo, the patient-derived xenograft model showed engraftment of APL samples upon MN1-OE, with the colonization of secondary organs and splenomegaly, which was not observed in the control. Flow cytometry analysis revealed that MN1-engrafted mice displayed increased levels of APL blasts in comparison with control, which had a large proportion of mature myeloid cells (CD33+CD11b+). Ex vivo screen of engrafted cells revealed increased resistance to ATRA-induced differentiation in MN1-OE sorted blasts. We provide further evidence to support the role of MN1 as an oncogenic hit allowing in vivo studies of difficult-to-engraft AML subtypes. Functionally, high MN1 expression resulted in decreased differentiation, retention of stem-like properties, and metabolic shift towards a more glycolytic state. We demonstrated that DNAJB6b controls the cellular localization of MN1, with its downregulation associated with the loss of MN1 supportive functions. Finally, our findings suggest that targeting glycolysis can be leveraged as a therapeutically actionable mechanism for driving cell death in MN1high AMLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of Polo-like Kinase 4 Induced Both Cell Intrinsic and Non-Cell Intrinsic Anti-Leukemia Effects on TP53-Mutated Acute Myeloid Leukemia

Inhibition of Polo-like Kinase 4 Induced Both Cell Intrinsic and Non-Cell Intrinsic Anti-Leukemia Effects on TP53-Mutated Acute Myeloid Leukemia

Optimal Induction and Consolidation Strategy for Patients with Complex Karyotype and TP53-Mutated Acute Myeloid Leukemia

Optimal Induction and Consolidation Strategy for Patients with Complex Karyotype and TP53-Mutated Acute Myeloid Leukemia

Impact of Allelic State on Overall Survival in TP53-mutant Acute Myeloid Leukemia (AML) and Higher Risk Myelodysplastic Syndromes (HR-MDS)

Introduction: Mutations in TP53 confer an adverse prognosis in patients (pts) with AML and HR-MDS. There are conflicting results regarding the prognostic impact of mono-allelic vs bi-allelic and mono-hit vs multi-hit TP53 mutation status on prognosis in AML and HR-MDS. Additionally, assessment of loss-of-heterozygosity (LOH) status is not routinely available in many settings limiting the clinical implementation of TP53 mutational status. Methods: We took advantage of a large, randomized study with extensive clinical and molecular annotation to analyze 61 patients (pts) with TP53-mutated (TP53-mut) disease and to compare them to 144 TP53 wild-type pts. All pts received azacitidine and half were randomized to receive the anti-PDL1 antibody durvalumab, which did not improve clinical outcomes (Zeidan et al. Blood Advances 2022). A 38 gene-targeted myeloid mutation analysis from screening bone marrow was performed at Munich Leukemia Laboratory. Only pathogenic TP53 variants with variant allele frequency (VAF) ≥2% were included. Pts with a common SNP or VUS in the TP53 gene were classified as TP53-WT in the analysis. Multi-hit TP53 mutations were defined as previously published by the International Consensus Conference (ICC; Arber et al. Blood 2022) as ≥2 distinct TP53 mutations (VAF ≥10%) or a single TP53 mutation associated with either: 1) cytogenetic deletion involving chromosome 17p (e.g., del(17p) or monosomy 17); 2) a VAF of >50%; or 3) with any complex karyotype. We also defined multi-hit TP53 mutations as proposed by Grob et al. (Blood 2022) as (I) ≥2 TP53 gene variants irrespective of VAF, (II) ≥1 TP53 gene variant co-occurred with a cytogenetic deletion involving chromosome 17p, or (III) TP53 mutations with VAF >55%. Based on multiple prior studies (Grob et al. Blood 2022, Weinberg et al. Blood Advances 2022) patients with AML and HR-MDS with TP53 mutations were analyzed as one disease entity. Results: 37 had TP53-mut AML, 89 were TP53-WT AML, 24 had TP53-mut HR-MDS, and 55 were TP53-WT HR-MDS pts. The average VAF for TP53 mutations was 37%, and 90% had ≥10% VAF. 93% of TP53 mutations occurred in the p53 DNA-binding domain. TP53-mut pts had fewer co-occurring mutations compared with TP53-WT pts (mean number of mutations: 1.8 [SD: 0.77] for TP53-mut pts (including TP53) vs 2.9 [SD: 1.4] for TP53-WT pts; Wilcoxon P <0.001) with distinct patterns in the distribution of co-occurring mutations. In terms of TP53 allelic status, 22 (11.0%) were mono-hit and 41 (20.5%) pts were multi-hit as defined by Grob et al, while 7 (3.4%) and 56 (28.0%) pts were classified as mono-hit and multi-hit per ICC definition, respectively. In the AML cohort, TP53-mut pts were more likely to have poor-risk cytogenetics (59.5% vs 11.2%; p<0.001), therapy-related myeloid neoplasm (10.8% vs 1.1%; p=0.042), and lower bone marrow blast percentage (27.5% vs 35.5%; p=0.001) compared to TP53-WT. Among MDS pts, TP53-mut pts were more likely to have secondary MDS (20.8% vs 1.8%; p=0.013), very poor risk cytogenetics by IPSS-R (75% vs 14.6%; p<0.001), and very high risk IPSS-R score (62.5% vs 34.6%; p=0.039). There were no statistically significant differences in terms of baseline demographics and other clinical characteristics. There was no difference in overall response rate (ORR) between TP53-mut and TP53-WT pts (AML: ORR: 35.1% [95% CI: 20.7% - 52.6%] vs. 33.7% [95% CI: 24.2% - 44.6%]; p=1.000; MDS: ORR: 41.7% [95% CI: 22.8% - 63.1%] vs. 60.0% [95% CI: 45.9% - 72.7%]; p=0.208). TP53 allelic status did not influence OS using either definition (Grob et al: mono-hit: median OS 7.6 months [95% CI:5.4-10.6 months]; multi-hit 10.6 months [95% CI: 9.1-13.0 months] (Figure A); ICC definition: 8.0 months (95% CI: 6.1-20+ months) and 9.5 months [95% CI: 7.7-10.9 months], (Figure B)) and was inferior to TP53-WT (median OS: 18.9 months; 95% CI: 15.5-22.9 months). Conclusion: Outcomes of AML and MDS pts with TP53 mutations are worse compared to TP53-WT without differences between mono-hit vs multi-hit status as defined by the ICC and Grob et al. This suggests that either the negative prognostic impact of a multi-hit TP53 allelic status can be overcome by treatment with azacitidine or that a distinction by TP53 allelic status in newly diagnosed AML/HR-MDS pts treated with azacitidine does not provide additional prognostic information. Whether incorporating TP53 LOH status is necessary to further refine risk assessment warrants additional studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictors of Long-Term Outcome in TP53-Mutated Acute Myeloid Leukemia Patients Receiving Allogeneic Stem Cell Transplant after First- or Second-Line Therapy: Results from the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND)

Predictors of Long-Term Outcome in TP53-Mutated Acute Myeloid Leukemia Patients Receiving Allogeneic Stem Cell Transplant after First- or Second-Line Therapy: Results from the Consortium on Myeloid Malignancies and Neoplastic Diseases (COMMAND)

AML-464 Tolerability and Efficacy of the First-In-Class Anti-CD47 Antibody Magrolimab Combined With Azacitidine in Frontline Patients With TP53-Mutated Acute Myeloid Leukemia (AML): Phase 1b Results

Patients with TP53-mutated AML have a poor prognosis. Magrolimab is an antibody blocking CD47, a "don't eat me" signal on cancer cells, which induces tumor phagocytosis and is synergistic with azacitidine. Report final tolerability and efficacy data. Ph1b single-arm trial of magrolimab+azacitidine (NCT03248479). 72 frontline patients with TP53-mutated AML unsuitable for intensive chemotherapy. Magrolimab IV 1-mg/kg (priming) then 30-mg/kg ramp-up QW/Q2W (maintenance). Azacitidine 75 mg/m2 IV/SC days 1-7 (each 28-day cycle). Primary endpoints were safety/tolerability and complete remission (CR) rate. Common treatment-emergent adverse events (TEAEs) were constipation (52.8%), diarrhea (47.2%), febrile neutropenia (45.8%), nausea (43.1%), fatigue (37.5%), decreased appetite (37.5%), thrombocytopenia (31.9%), peripheral edema (30.6%), and cough (30.6%). Common grade (G)≥3 TEAEs were febrile neutropenia (37.5%), anemia (29.2%; G3, 26.4%; G4, 2.8%), thrombocytopenia (29.2%), pneumonia (26.4%), and neutropenia (20.8%). G3 hemolysis was reported in 1 patient; no G4 hemolysis was reported. Objective response rate was 48.6% (CR, 33.3%; CR with incomplete hematologic recovery [CRi]/CR with partial hematologic recovery [CRh], 8.3%; morphologic leukemia-free state [MLFS], 1.4%; partial remission, 5.6%). 16.7% and 5.6% of patients had stable disease and progressive disease (PD), respectively; 30- and 60-day mortality rates were 8.3% and 18.1%, respectively. Response assessments were unavailable in 4.2% (discontinued due to AEs) and 6.9% (other) of patients prior to the C3D1 assessment. Median time to CR/CRi was 2.2 months (range, 1.7-7.2) and CR was 3.0 months (range, 1.8-9.6); 14/31 (45.2%) evaluable patients with CR/CRi/CRh/MLFS achieved negative MRD. 8/24 patients with CR had a longitudinal TP53 variant-allele-frequency (VAF) assessment; 5/8 (63%) had ≤5% VAF. Treatment was stopped due to stem cell transplant (9 [12.5%]), PD (26 [36.1%]), death (8 [11.1%]), AE (13 [18.1%]), or other (14 [19.4%]). Median durations of CR and CR/CRi were 7.7 (95% CI, 4.7-10.9) and 8.7 (95% CI, 5.3-10.9) months, respectively. Median overall survival was 10.8 months (95% CI, 6.8-12.8; 8.3-month median follow-up). Magrolimab+azacitidine showed durable responses and encouraging overall survival in frontline patients with TP53-mutated AML unsuitable for intensive chemotherapy. A Ph3 trial of magrolimab+azacitidine vs standard-of-care in TP53-mutated AML (ENHANCE-2; NCT04778397) is ongoing.

AML-191 A Single Institution Experience on Clinical Outcomes of TP-53 Mutant Acute Myeloid Leukemia (AML)

TP53 (tumor suppressor gene, 'the guardian of the genome') regulates DNA repair and cell division. TP53 mutations are observed in up to 20% of AML patients. It carries an adverse prognosis and is associated with inferior outcomes. Analyze TP53 mutations impact in AML patients. We reviewed patients with TP53 mutated AML treated at our institution. All cases of TP53 mutated AML (9 patients, 6.9%) of 130 AML patients were included. Nine patients were identified with newly diagnosed TP53 mutated AML. Fit non-APL patients received induction chemotherapy (IC) with purine analogues, cytarabine-anthracycline. Unfit patients received intensity chemotherapy (LIC) with a hypomethylating agent and venetoclax or cladribine, low dose cytarabine (LDAC) and venetoclax. Complete response (CR) rate and overall survival (OS) were analyzed. Among 130 patients with AML, we identified with TP53 mutations. 5(55%) were women. Races included Whites (n=2), Blacks (n=5) and Hispanics (n=2). Median age was 66 years (range, 41-80). Eight patients (88%) had complex cytogenetics. A coexisting IDH1 and DNMT3A mutation were present in 2 and 1 patient, respectively. 2 patients received FIA, and none achieved a CR. One patient treated with FIA developed severe neutropenic typhlitis and multiorgan failure. Unfit patients received HMA alone (n=2), HMA+ Ven (n=3), Cladribine, LDAC and Ven (n=2). Among 3 patients treated with HMA plus Ven; 2 achieved a CR. 2 patients treated with cladribine, LDAC, and Ven achieved CR after first cycle, and both remain in CR at a mean follow-up of 5 months. None received allogenic stem cell transplant (ASCT) in CR 1, but one patient underwent ASCT in CR 2 and died 2 months after transplant from graft failure and infection. Median OS in the entire cohort was 180 days (95% CI. 39.0-393.0 days). Our analysis though small in numbers seems to confirm the poor response to intense cytotoxic chemotherapy in TP53 mutant AML as described in recent medical literature (Short et al., Blood Advances, 2022). LIC is well tolerated and can be used as an effective bridging strategy to ASCT.

How I Treat TP53-Mutated Acute Myeloid Leukemia and Myelodysplastic Syndromes.

Simple SummaryTP53 mutations are seen in 5–10% of de novo MDS and AML, but 25–40% of therapy-related MDS and AML. Despite the addition of recent drugs to the common regimen and improvement of post transplantation survival, these particular myeloid malignancy subtypes remain a challenge for hematologists around the globe. In this article, we aim at reviewing the biology and most recent advances in the treatment of TP53 MDS and AML. TP53-mutated acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are among the myeloid malignancies with the poorest prognosis. In this review, we analyze the prognosis of these two diseases, focussing particularly on the extent of the mono or biallelic mutation status of TP53 mutation, which is largely correlated with cytogenetic complexity. We discuss the possible/potential improvement in outcome based on recent results obtained with new drugs (especially eprenetapopt and magrolimab). We also focus on the impact of allogeneic hematopoietic stem cell transplantation (aHSCT) including post aHSCT treatment.

Outcomes with allogeneic hematopoietic stem cell transplantation in TP53-mutated acute myeloid leukemia: a systematic review and meta-analysis

We conducted a systematic review and meta-analysis to evaluate outcomes after allogeneic hematopoietic stem cell transplantation (HSCT) in TP53-mutated acute myeloid leukemia (AML). We performed a literature search on PubMed, Embase, Cochrane Library, and ClinicalTrials.gov. After screening 592 manuscripts, eight studies were included. Data were extracted following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Pooled analysis was done using the meta-package by Schwarzer et al. Proportions with 95% confidence intervals (CIs) were computed. We analyzed 297 patients. The median follow-up was 45 (0.9–407.3) months. The pooled 2-year overall survival was 29.7% (95% CI 0.17–0.43, n = 82/248). The pooled relapse rate was 61.4% (95% CI 0.41–0.79, n = 139/247) at a median follow-up time of 2 (0.26–3) years. Three-year progression-free survival and non-relapse mortality were reported by one study as 7.5% and 32.5%, respectively. Outcomes of HSCT for TP53-mutated AML are poor; however, HSCT confers a survival advantage as compared to non-transplant palliative therapies.

S132: TOLERABILITY AND EFFICACY OF THE FIRST-IN-CLASS ANTI-CD47 ANTIBODY MAGROLIMAB COMBINED WITH AZACITIDINE IN FRONTLINE PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA: PHASE 1B RESULTS

Background: Patients (pts) with TP53-mutated acute myeloid leukemia (AML) have a poor prognosis, with limited responses to currently available therapies and low survival outcomes, representing a significant unmet medical need. Magrolimab is a monoclonal antibody blocking CD47, a “don’t eat me” signal overexpressed on cells in cancers such as AML. This blockade induces phagocytosis of tumor cells and is synergistic with azacitidine (AZA) via upregulation of “eat me” signals. Aims: To report tolerability and efficacy data from a phase 1b trial of magrolimab + AZA in frontline pts with TP53-mutated AML unsuitable for intensive chemotherapy (NCT03248479). Methods: Frontline pts with AML not suitable for intensive chemotherapy received magrolimab IV starting with a priming dose (1 mg/kg) followed by ramp-up to 30 mg/kg QW or Q2W as the maintenance dose. AZA 75 mg/m2 was given IV or SC on days 1-7 of each 28-day cycle. Primary endpoints were safety/tolerability and complete remission (CR) rate by European LeukemiaNet (ELN) 2017 criteria. Results: 72 pts with TP53-mutated AML were treated (Table). Common all-grade treatment-emergent adverse events (TEAEs) were constipation (52.8%), diarrhea (47.2%), febrile neutropenia (45.8%), nausea (43.1%), fatigue (37.5%), decreased appetite (37.5%), thrombocytopenia (31.9%), peripheral edema (30.6%), and cough (30.6%). Most common grade ≥3 TEAEs were febrile neutropenia (37.5%), anemia (29.2%; grade 3, 26.4%; grade 4, 2.8%), thrombocytopenia (29.2%), pneumonia (26.4%), and neutropenia (20.8%). Grade 3 hemolysis was reported in 1 pt (1.4%); no grade 4 hemolysis was reported. Objective response rate by intent to treat was 48.6% (CR, 33.3%; CR with incomplete hematologic recovery [CRi]/CR with partial hematologic recovery [CRh], 8.3%; morphologic leukemia-free state [MLFS], 1.4%; partial remission, 5.6%). Stable disease and progressive disease (PD) were reported in 16.7% and 5.6% of pts, respectively; 30- and 60-day mortality rates were 8.3% and 18.1%, respectively. Response assessments were unavailable in an additional 4.2% of pts who discontinued due to AEs and 6.9% due to other reasons, prior to the cycle 3 day 1 assessment. Median time to CR/CRi was 2.2 months (range, 1.7-7.2 months) and to CR was 3.0 months (range, 1.8-9.6 months); 14 of 31 (45.2%) evaluable pts with CR/CRi/CRh/MLFS achieved negative MRD by flow cytometry (investigator reported). Of 24 pts with CR, 8 had a longitudinal TP53 variant allele frequency (VAF) assessment and 5 of 8 (63%) had VAF decreased to ≤5%. Treatment was stopped due to stem cell transplant (9 [12.5%]), PD (26 [36.1%]), death (8 [11.1%]), AE (13 [18.1%]), and other (14 [19.4%]). Median durations of CR and CR/CRi were 7.7 months (95% CI, 4.7-10.9 months) and 8.7 months (95% CI, 5.3-10.9 months), respectively. Median overall survival (OS) in 72 pts was 10.8 months (95% CI, 6.8-12.8 months) (figure), with median follow-up of 8.3 months. - Table. Baseline characteristics N=72 Age (range), years 73 (31-89) ECOG, n (%) 0-1 61 (84.7) 2 11 (15.3) ELN cytogenetic risk, n (%) Favorable 1 (1.4) Intermediate 2 (2.8) Adverse 57 (79.2) Unknown 12 (16.7) AML with MDS-related changes, n (%) 34 (47.2) Therapy-related AML, n (%) 15 (20.8) ECOG, Eastern Cooperative Oncology Group; MDS, myelodysplastic syndrome. Image:Summary/Conclusion: In high-risk frontline pts with TP53-mutated AML unsuitable for intensive chemotherapy, magrolimab + AZA showed durable responses and encouraging OS in a single-arm study. A Phase 3 trial of this combination vs standard of care in TP53-mutated AML (ENHANCE-2; NCT04778397) is ongoing.

PB1820: CD47-BLOCKER TTI-622 COMBINED WITH AZACITIDINE IN PATIENTS WITH TP53-MUTATED ACUTE MYELOID LEUKEMIA (AML) AND WITH AZACITIDINE + VENETOCLAX IN ELDERLY OR UNFIT PATIENTS WITH TP53-WILDTYPE AML

Background: CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a “don’t eat me” signal to suppress macrophage phagocytosis. TTI-622 is a fusion protein consisting of the CD47-binding domain of human SIRPα linked to the Fc region of human IgG4. It is designed to enhance phagocytosis and antitumor activity by preventing CD47 from delivering its inhibitory signal as well as generating a moderate pro-phagocytic signal via IgG4 Fc. Importantly, unlike many CD47-blocking agents, TTI-622 does not bind to human red blood cells. Increased expression of CD47 is an adverse prognostic factor in acute myeloid leukemia (AML) due to its association with reduced event-free and overall survival, and blockade of CD47 has shown pre-clinical and clinical activity in AML. In vitro, both azacitidine and venetoclax significantly enhanced the pro-phagocytic effect of TTI-622 on macrophages. Aims: To characterize the safety and preliminary efficacy profile of TTI-622 in combination with azacitidine in newly diagnosed TP53-mutated AML and in combination with azacitidine + venetoclax in elderly or unfit, newly diagnosed TP53-wildtype AML. Pharmacokinetics and immunogenicity of TTI-622 combined with azacitidine ± venetoclax will also be investigated. Primary endpoints include dose-limiting toxicities, incidence of adverse events (AEs) and complete response (CR). Minimal residual disease status will also be evaluated. Methods: As part of an ongoing dose-escalation and expansion trial of TTI-622 (NCT03530683), patients with AML meeting the below key entry criterion are enrolled. Cohort A includes patients with newly diagnosed TP53-mutated AML. Cohort B includes patients with newly diagnosed TP53-wildtype AML who are either elderly (375 years) or unfit. Unfit patients include those < 75 years with one or more of the following comorbidities: Eastern Cooperative Oncology Group (ECOG) performance status of 2, clinically significant heart or lung comorbidities, contraindication to anthracycline therapy, or other comorbidity the investigator regards as incompatible with intensive remission induction chemotherapy. All patients from both cohorts must have documented bone marrow or peripheral blast counts 3 20% and must have received no prior systemic therapy for AML nor hypomethylating agents for any antecedent hematologic disorders prior to transformation to AML. Patients must have no extramedullary disease or leukocytosis >25,000/mL. Renal function must be 330 mL/minute based on Cockcroft-Gault estimate. There are no restrictions based on neutrophil or platelet counts, or hemoglobin level. Patients with AML secondary to antecedent hematologic disorders or therapy induced will not comprise more than 50% of either Cohort. Up to 30 patients will be enrolled into each cohort. In 28-day cycles, patients in both Cohorts will receive TTI-622 8 mg/kg intravenous (IV) infusions weekly combined with azacitidine 75 mg/m2 administered either IV or subcutaneously for 7 consecutive days, repeated approximately every 4 weeks. Patients in Cohort B will also receive venetoclax 100 mg and 200 mg orally on Days 1 and 2, respectively, followed by 400 mg (or equivalent adjusted for azoles) daily thereafter. Safety monitoring includes clinical laboratories and assessments of AEs based on CTCAE v 4.03. Response assessments of both bone marrow and peripheral blood are performed at the end of Cycles 1, 2 and 4; and then every 3 cycles thereafter. Response assessment is per the ELN2017 criteria. Results: Results N/A (trial-in-progress) Summary/Conclusion: Conclusion N/A (trial-in-progress)

Abstract 6301: DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML

Abstract DNA methyltransferase inhibitors (DNMTis), which transcriptionally activate hypermethylated genes in cancers and leukemias, also activate endogenous retroviruses (ERVs), leading to increased cytosolic double-stranded (ds) RNA and interferon (IFN) signaling, in a process termed viral mimicry. The tumor suppressor TP53 has been reported to cooperate with DNA methylation and IFN signaling to maintain transcriptional silencing of various repeat sequences, including ERVs. We now report that DNMTis, used to treat acute myeloid leukemia (AML) patients unfit for chemotherapy, robustly induce expression of ERVs and STING-dependent IFN/inflammasome signaling in AML with mutations in TP53(approximately 10%), a subset with poor prognosis. First, our studies of ERV transcripts (N=13) levels in TP53 mutant (KASUMI, KG1-1A, U937) and wild-type (WT; MOLM-14, OCI/AML2, and OCI/AML3) AML cell lines and primary cells (N=6 mutant, N=6 WT) show that, while TP53 mutant cells have low baseline ERV expression, DNMTi treatment significantly increases ERV expression, compared with WT TP53 cells (p&amp;lt;0.05). Moreover, inhibiting TP53 in WT TP53 AML cell lines with the specific TP53 inhibitor pifithrin significantly upregulates ERVs, validating the role of TP53 in suppressing ERVs in AML cells. Second, while TCGA data analysis showed that baseline STING transcript are low in TP53 mutant vs WT AML, phospho-STING (activated) is actually increased in TP53 mutant AML cells and DNMTi treatment further activates STING. Increased expression of downstream IFN/inflammasome genes in TP53 mutant AML treated with DNMTi, compared with results in WT cells, validates these results. Moreover, DNMTi combination with poly (ADP-ribose) polymerase inhibitors (PARPis), which we previously reported to activate STING in breast and ovarian cancer though increasing cytosolic dsDNA and dsRNA, further augments STING activation and IFN/inflammasome signaling (p&amp;lt;0.05). Third, the IFN/inflammasome signaling response to DNMTI/PARPI signaling is directly linked with decreased expression of DNA ds break repair (DSBR) genes and activity, leading to homologous recombination defects (HRD). Fourth, the mechanism for both IFN/inflammasome signaling and HRD induction is dependent on STING, since both CRISPR/CAS KO of STING or treatment with the STING inhibitor H-151 can rescue these effects in TP53 mutant and WT AML cells. Our findings increase understanding of the therapy potential for combining DNMTi+PARPi treatment in AML and suggest use of this paradigm to treat mutant TP53 AML. These results also suggest that activating STING may be a key strategy for increasing IFN/inflammasome signaling in treatment of TP53 mutant AML and that immune responses may be further augmented with immune therapy in AML and other cancers. Citation Format: Aksinija A. Kogan, Michael J. Topper, Lora Stojanovic, Lena J. McLaughlin, Tammy J. Kingsbury, Maria R. Baer, Michael Kessler, Stephen B. Baylin, Feyruz V. Rassool. DNA methyltransferase inhibitors increase ERV reactivation and STING-dependent interferon/inflammasome signaling in TP53 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6301.