TP53-mutant Cells Research Articles

Targeting ATR in TP53-Mutant AML/MDS

Mutations of TP53 are found in approximately 10% of cases of de novo AML/MDS and approximately 30% of therapy related AML/MDS. These patients have an exceptionally poor prognosis, with median survival of 6-9 months. Many patients with TP53-mutant AML/MDS respond to hypomethylating agents, although the response is not durable, similar to standard induction therapy. The mechanisms that contribute to the relative sensitivity of TP53-mutated AML/MDS to hypomethylating agents is unknown. Hypomethylating agents work, at least in part, by inhibiting DNMT1, the major DNA methyltransferase responsible for maintaining methylation of genomic DNA. Loss of DNMT1 and DNA hypomethylation has been previously shown to induce DNA replication stress and activate the ATM-Rad3-related (ATR) pathway. TP53 has multiple roles in the replication stress response, including control of the G1/S checkpoint and regulation of DNA replication progression and restart. Based on these observations, we hypothesize that ATR inhibition selectively sensitizes TP53-mutated MDS/AML to decitabine-induced replication stress. To test this hypothesis, initial studies focused on MOLM13 cells, a TP53-wildtype human AML cell line. Treatment with decitabine for 4 days induced global hypomethylation, and increased phosphorylation of CHK1, which is indicative of ATR activation. We next performed single-molecule DNA fiber assays to assess replication fork progression after exposure to decitabine. We used CRISPR-Cas9 gene editing to generate TP53-/- (or control) MOLM13 cells. In untreated cells, no difference in replication fork progression rate was observed between wildtype and TP53-/- MOLM13 cells (Figure 1). However, after decitabine treatment, a significant decrease in replication fork length was observed in TP53-/- MOLM13, but not wildtype MOLM13 cells. These data show that p53 is required for the efficient resolution of replication stress induced by decitabine. Since ATR is a major regulator of replication stress, we next examined the response of TP53-/- MOLM13 cells to decitabine alone, AZD6738 (an ATR inhibitor) alone, or the combination. Compared to controls, TP53-/- MOLM13 cells showed a modest increased sensitivity to decitabine, but not AZD6738. However, strong synergistic killing was observed with the combination in TP53-/-, but not control, MOLM13 cells (Zip synergy score of 10.1 vs 5.8). Similar results were observed in a second cell line, murine AML1. Cell cycle analysis showed that combined treatment results in a selective increase in polyploid TP53-/- MOLM13 cells, which is suggestive of mitotic catastrophe. We next developed a xenotransplantation model of TP53-mutated AML by transplanting TP53-/- or control MOLM13 cells into NSG mice. After 4 days to allow for engraftment, mice were treated with vehicle alone, decitabine alone (0.1mg/kg), AZD6738 alone (50mg/kg), or the combination for 14 days. In mice engrafted with wildtype MOLM13 cells, no significant benefit of single agent or combination therapy was observed. In contrast, treatment with decitabine alone modestly, but significantly, prolonged survival in mice engrafted with TP53-/- MOLM13 cells (Figure 2). Importantly, the survival of mice treated with combined AZD6738 and decitabine had a significantly prolonged survival compared to decitabine alone. These data show that even a single cycle of AZD6738 and decitabine is sufficient to prolong survival in the xenograft model of human AML. Finally, to assess the impact of combination treatment on TP53-mutant hematopoietic stem cells (HSCs), we established mixed bone marrow chimeras transplanted with both wildtype and R172H TP53 bone marrow. Mice were treated with either cis-platinum or decitabine ± AZD6738. Whereas cis-platinum treatment resulted in a significant expansion of TP53-mutant HSCs, treatment with decitabine plus AZD6738 selectively inhibited TP53-mutant HSCs. Collectively, these data support the hypothesis that TP53-mutated AML/MDS is selectively sensitive to ATR inhibition in combination with decitabine-induced replicative stress. A clinical trial of this drug combination in patients with TP53-mutated AML/MDS is being developed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

265. SIGNIFICANCE OF AGR2 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Abstract Anterior gradient 2 (AGR2) promotes tumor growth and make a worse prognosis in several cancers, especially in adenocarcinoma. However, comprehensive functional analysis of AGR2 in esophageal squamous cell carcinoma (ESCC) has not been reported. In the present study, the functional analysis and clinical significance of AGR2 were examined using ESCC cell lines and clinical samples. AGR2 was upregulated in ESCC cell lines and ESCC tissue sample. The downregulation of AGR2 suppressed cell proliferation and increased the proportion of G2/M-phase cells. Furthermore, phosphorylation of p53 in TP53-wild-type ESCC and osteosarcoma cells was also promoted. However, these changes were not observed in TP53-mutant ESCC cells. Immunohistochemistry results showed that high AGR2 and low p53 expression in ESCC tissue was correlated with a worse prognosis. These results suggest that although AGR2 enhances cell proliferation by inhibiting p53 phosphorylation in TP53-wild-type ESCC, the same mechanism does not regulate cell functions in TP53-mutant ESCC due to suppression of TP-53 function. In conclusion, AGR2 plays an important role in ESCC progression and might be a useful prognostic marker in patients with TP53-wild-type ESCC.

Multi-Omic Analysis of Two Common P53 Mutations: Proteins Regulated by Mutated P53 as Potential Targets for Immunotherapy.

Simple SummaryTP53 is the most frequently mutated gene in many cancers, but it has failed to be a very effective target for treatment to date. To overcome this, we have examined what else changes in cells when the TP53 gene is mutated. We modified cells that had no TP53 expression to have one of the two most common mutations, either R175H or R273H. We examined how the presence of these TP53 mutations caused cellular changes including microscopic, gene expression and peptide presentation to the immune system. This has allowed us to identify new (secondary) targets that could be used to facilitate the treatment of tumors that harbor p53 mutations.The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein–protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.

Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Quantifying Cellular Repair, Misrepair and Apoptosis Induced by Boron Ions, Gamma Rays and PRIMA-1 Using the RHR Formulation

The recent interaction cross-section-based formulation for radiation-induced direct cellular inactivation, mild and severe sublethal damage, DNA-repair and cell survival have been developed to accurately describe cellular repair, misrepair and apoptosis in TP53 wild-type and mutant cells. The principal idea of this new non-homologous repairable-homologous repairable (RHR) damage formulation is to separately describe the mild damage that can be rapidly handled by the most basic repair processes including the non-homologous end joining (NHEJ), and more complex damage requiring longer repair times and high-fidelity homologous recombination (HR) repair. Taking the interaction between these two key mammalian DNA repair processes more accurately into account has significantly improved the method as indicated in the original publication. Based on the principal mechanisms of 7 repair and 8 misrepair processes presently derived, it has been possible to quite accurately describe the probability that some of these repair processes when unsuccessful can induce cellular apoptosis with increasing doses of γrays, boron ions and PRIMA-1. Interestingly, for all LETs studied (≈0.3-160 eV/nm) the increase in apoptosis saturates when the cell survival reaches about 10% and the fraction of un-hit cells is well below the 1% level. It is shown that most of the early cell kill for low-to-medium LETs are due to apoptosis since the cell survival as well as the non-apoptotic cells agree very well at low doses and other death processes dominate beyond D > 1 Gy. The low-dose apoptosis is due to the fact that the full activation of the checkpoint kinases ATM and Chk2 requires >8 and >18 DSBs per cell to phosphorylate p53 at serine 15 and 20. Therefore, DNA repair is not fully activated until well after 1/2 Gy, and the cellular response may be apoptotic by default before the low-dose hyper sensitivity (LDHS) is replaced by an increased radiation tolerance as the DNA repair processes get maximal efficiency. In effect, simultaneously explaining the LDHS and inverse dose rate phenomena. The partial contributions by the eight newly derived misrepair processes was determined so they together accurately described the experimental apoptosis induction data for γ rays and boron ions. Through these partial misrepair contributions it was possible to predict the apoptotic response based solely on carefully analyzed cell survival data, demonstrating the usefulness of an accurate DNA repair-based cell survival approach. The peak relative biological effectiveness (RBE) of the boron ions was 3.5 at 160 eV/nm whereas the analogous peak relative apoptotic effectiveness (RAE) was 3.4 but at 40 eV/nm indicating the clinical value of the lower LET light ions (15 ≤ LET ≤ 55 eV/nm, 2 ≤ Z ≤5) in therapeutic applications to maximize tumor apoptosis and senescence. The new survival expressions were also applied on mouse embryonic fibroblasts with key knocked-out repair genes, showing a good agreement between the principal non-homologous and homologous repair terms and also a reasonable prediction of the associated apoptotic induction. Finally, the formulation was used to estimate the increase in DNA repair and apoptotic response in combination with the mutant p53 reactivating compound PRIMA-1 and γ rays, indicating a 10-2 times increase in apoptosis with 5 µM of the compound reaching apoptosis levels not far from peak apoptosis boron ions in a TP53 mutant cell line. To utilize PRIMA-1 induced apoptosis and cellular sensitization for reactive oxygen species (ROS), concomitant biologically optimized radiation therapy is proposed to maximize the complication free tumor cure for the multitude of TP53 mutant tumors seen in the clinic. The experimental data also indicated the clinically very important high-absorbed dose ROS effect of PRIMA-1.

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<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<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.

Abstract 5593: DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity

Abstract DNA methyltransferase inhibitors (DNMTis) modulate genome-wide methylation patterns, previously linked by our group to downregulation of DNA double strand break repair (DSBR) that sensitizes non-small cell lung cancer (NSCLC) to poly(ADP-ribose) polymerase inhibitors (PARPis). DNMTi hypomethylation of endogenous retroviral (ERV) elements produces cytosolic aggregation of double-stranded RNA (dsRNA) that activates antiviral interferon (IFN) and inflammasome signaling. Accordingly, DNMTi treatment of NSCLC cell lines (A549, H460) increases expression of 1) multiple ERV transcripts; 2) dsRNA sensors RIG-I and MDA5; 3) downstream mitochondrial antiviral signaling protein (MAVS); 4) MAVS-activated antiviral kinases (TBK1, IKKα/β) and transcriptional regulators (IRF3/7, NFκB); and 5) IFNβ and IFN-stimulated genes (ISGs). A second mechanism of cellular antiviral signaling is STING (stimulator of interferon genes) activation triggered by cytosolic dsDNA. Following DNMTi treatment, we observe mitochondrial dysfunction including impairment of oxidative phosphorylation, reactive oxygen species generation, and reduced mitochondrial membrane potential. This dysfunction is associated with increased mitochondrial DNA (mtDNA) damage and accumulation of cytosolic mtDNA, leading to increased STING expression and IFN activation that can be abrogated by siSTING or IKK inhibition. Treatment with PARPi, which activates STING via cytosolic accumulation of damaged nuclear DNA, further enhances DNMTi-induced IFN signaling. We previously linked IFN activation to DSBR downregulation in breast and ovarian cancer, and now present TCGA analysis in NSCLC indicating a similar negative correlation, corroborated by reduced DSBR in NSCLC cell lines after IFNβ treatment. Computational interrogation indicates multiple DSBR promoters contain IFN-stimulated response elements (ISREs), including potential negative regulators, that may account for these findings. Notably, we find that precise patterns of immune activation may be TP53 dependent: NSCLC cell lines with missense mutations (H23, H441, H1115), as observed in ~30% patients, exhibit increased NFκB inflammasome signaling and decreased IFN activation compared to wildtype (A549, H460). TP53 mutation is associated with significantly reduced baseline STING expression, potentially affording a more robust DNMTi treatment response; accordingly, TP53-mutant NSCLC cells exhibit significantly greater sensitivity to DNMTi/PARPi. Our new data expands on our published work to further understanding of novel mechanisms of 1) inflammasome induction and 2) mitochondrial dysfunction through which DNMTis activate cellular antiviral signaling and downregulate DSBR to induce PARPi sensitivity in NSCLC. We also identify TP53 as a possible biomarker for future personalization of DNMTi therapy. Citation Format: Rachel Abbotts, Lora Stojanovic, Michael J. Topper, Brian M. Polster, Stephen B. Baylin, Feyruz V. Rassool. DNMT inhibitor induces NSCLC inflammasome signaling and mitochondrial dysfunction, producing to DSB repair defects and PARP inhibitor sensitivity [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 5593.

Abstract 5439: MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma

Abstract Multiple myeloma (MM) accounts for about 2% of all cancers and 18% of all hematologic malignancies in the US. Newly developed treatments (e.g., immunomodulators, proteasome inhibitors, monoclonal antibodies) have significantly improved outcomes, but MM inevitably relapses and is considered incurable. Genomic analysis shows that the TP53 gene encoding tumor suppressor protein p53 is infrequently mutated in patients with MM, of whom about 82% retain wild-type (WT) TP53. Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that inhibits p53 via proteasome degradation. Proteasome inhibitors might help to stabilize p53 and synergize with MDM2 inhibitors. Therefore, MDM2 inhibitors that activate p53 might constitute an attractive pharmacologic approach to MM. Alrizomadlin (APG-115) is an investigational, new small molecule targeting the p53/MDM2 interaction and is in clinical development for solid and hematologic cancers. This study aimed to evaluate whether alrizomadlin can potentiate the antitumor effects of proteasome inhibitors in MM. In cell-based antiproliferation studies, alrizomadlin demonstrated selective activity against WT TP53 MM cell lines, including MOLP-8, H929, and MM1S, with IC50 values of 0.495 ± 0.132 µM, 0.259 ± 0.251 µM, and 0.325 ± 0.105 µM, respectively. In contrast, alrizomadlin was inactive against TP53-mutant MM cell lines (IC50 >10 µM). In MM cell lines, APG-115 exhibited synergistic activity with anti-MM agents (e.g., carfilzomib, lenalidomide, melphalan, selinexor) and other targeted agents (e.g., panobinostat, bromodomain inhibitor OTX-015, tumor necrosis factor-related apoptotic-inducing ligand). In vivo studies showed that coadministration of alrizomadlin with proteasome inhibitor bortezomib or carfilzomib enhanced tumor regression (vs. single agents) in H929 xenograft models. As to mechanism, alrizomadlin likely disrupts MDM2/p53 interactions and induces accumulation of p53, MDM2, and downstream proteins 4 hours after treatment. The proteasome inhibitors induced dose-related increases in p53 protein expression 24 hours after treatment. The combination of carfilzomib with alrizomadlin further promoted accumulation of p53 and MDM2 and increased phosphorylation of p53. As a transcription factor, p53: (1) activated expression of p21 to cause cell cycle arrest (2) augmented downstream BCL-2-associated X protein (BAX) and p53-upregulated modulator of apoptosis (PUMA), (3) increased cleavage of caspase-3 and poly [ADP-ribose] polymerase 1 (PARP-1; hallmarks of apoptosis), and (4) increased apoptosis and associated antitumor effects. In conclusion, our results demonstrate that the combination of MDM2 inhibitor APG-115 and proteasome inhibitors have synergistic antitumor effects on MM tumors harboring WT TP53. These data lay a foundation for clinical trials of a new and novel therapeutic option for patients with refractory MM. Citation Format: Guangfeng Wang, Eric Liang, Chunyang Tang, Li Rui, Ping Min, Jing Lv, Yangfeng Ge, Fei Zhang, Lvcheng Wang, Jingjin Shang, Dajun Yang, Yifan Zhai. MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma [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 5439.

Abstract 2574: Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages

Abstract As a key regulator of its dual role in transcription and cell cycle progression, CDK7 has been determined to be an attractive therapeutic target in a variety of tumor types driven by cell cycle dysregulation and aberrant control of the transcriptional processes. Q901 is an irreversible small molecule inhibitor with extreme selectivity and potent inhibition activity for CDK7, which forms a covalent bond with C312 of the CDK7 C-terminal domain. Q901 demonstrated selective cytotoxic activities in a broad spectrum of solid tumor lineages. TP53 mutation could be a potential predictive marker for the Q901 response as TP53 wild-type cancer cells demonstrates a significantly increased cytotoxic response compared to TP53 mutant cells in the bioinformatic analysis. In addition, gene expression profiling analysis showed a dose-response increase of POLR2A expression upon Q901 treatment and a correlation with tumor growth inhibition rate, indicating that POLR2A might be a potential PD marker for the Q901-induced response. Taken together, these data support the clinical development of Q901 for solid tumor indications. Q901 is progressing to IND-enabling studies to support the start of Phase 1 oncology clinical trials in early 2022. Citation Format: Donghoon Yu, Yeejin Jeon, Seung-Joo Lee, Jaeseung Kim, Kiyean Nam. Q901; a highly selective covalent cdk7 inhibitor inducing substantial anti-tumor effect in a broad spectrum of solid tumor lineages [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 2574.

Single-cell analysis reveals selection of TP53-mutated clones after MDM2 inhibition

AbstractThe mechanisms of transformation of chronic myeloproliferative neoplasms (MPN) to leukemia are largely unknown, but TP53 mutations acquisition is considered a key event in this process. p53 is a main tumor suppressor, but mutations in this protein per se do not confer a proliferative advantage to the cells, and a selection process is needed for the expansion of mutant clones. MDM2 inhibitors may rescue normal p53 from degradation and have been evaluated in a variety of cancers with promising results. However, the impact of these drugs on TP53-mutated cells is underexplored. We report herein evidence of a direct effect of MDM2 inhibition on the selection of MPN patients' cells harboring TP53 mutations. To decipher whether these mutations can arise in a specific molecular context, we used a DNA single-cell approach to determine the clonal architecture of TP53-mutated cells. We observed that TP53 mutations are late events in MPN, mainly occurring in the driver clone, whereas clonal evolution frequently consists of sequential branching instead of linear consecutive acquisition of mutations in the same clone. At the single-cell level, the presence of additional mutations does not influence the selection of TP53 mutant cells by MDM2 inhibitor treatment. Also, we describe an in vitro test allowing to predict the emergence of TP53 mutated clones. Altogether, this is the first demonstration that a drug treatment can directly favor the emergence of TP53-mutated subclones in MPN.

3170 – SINGLE-CELL MULTI-OMICS RESOLVES THE EVOLUTION OF TP53-MUTANT LEUKEMIA

TP53 regulates self-renewal and quiescence of hematopoietic stem cells, and its disruption leads to the development of hematological malignancies. In myeloid neoplasms, TP53 mutations define a distinct clinical entity, associated with complex cytogenetics and dismal outcomes. Understanding the cellular and molecular framework through which TP53 mutation drives clonal evolution is a crucial step towards the design of rational therapeutic strategies. Here, we carry out TARGET-seq single-cell multi-omic analysis of haematopoietic stem/progenitor cells (HSPC) from patients with a myeloproliferative neoplasm who had transformed to TP53-mutant secondary acute myeloid leukaemia (sAML), a trackable model of TP53-driven clonal evolution. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type (WT) alleles and gain of multiple chromosomal abnormalities upon transformation. TP53-mutant leukaemia stem cells (LSC) were transcriptionally distinct from de novo AML, with evidence of inflammation-associated transcription and aberrant erythroid differentiation. We identified a TP53-mutant LSC signature which was strongly predictive of adverse outcome in both TP53-mutant (HR:3.4) and WT AML (HR:3.1). Finally, we demonstrate a hitherto unrecognised effect of chronic inflammation in promoting disease progression. Sustained inflammatory stimuli (pIpC) led to a 2.5-fold expansion of TP53-mutant cells in WT:TP53R172H/+ chimeras, whereas WT cells were depleted. This indicates that pro-inflammatory cues promote fitness advantage of TP53-mutant cells whilst suppressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis ofTP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia and of broader relevance to other cancer types. TP53 regulates self-renewal and quiescence of hematopoietic stem cells, and its disruption leads to the development of hematological malignancies. In myeloid neoplasms, TP53 mutations define a distinct clinical entity, associated with complex cytogenetics and dismal outcomes. Understanding the cellular and molecular framework through which TP53 mutation drives clonal evolution is a crucial step towards the design of rational therapeutic strategies. Here, we carry out TARGET-seq single-cell multi-omic analysis of haematopoietic stem/progenitor cells (HSPC) from patients with a myeloproliferative neoplasm who had transformed to TP53-mutant secondary acute myeloid leukaemia (sAML), a trackable model of TP53-driven clonal evolution. We invariably identified convergent clonal evolution leading to complete loss of TP53 wild-type (WT) alleles and gain of multiple chromosomal abnormalities upon transformation. TP53-mutant leukaemia stem cells (LSC) were transcriptionally distinct from de novo AML, with evidence of inflammation-associated transcription and aberrant erythroid differentiation. We identified a TP53-mutant LSC signature which was strongly predictive of adverse outcome in both TP53-mutant (HR:3.4) and WT AML (HR:3.1). Finally, we demonstrate a hitherto unrecognised effect of chronic inflammation in promoting disease progression. Sustained inflammatory stimuli (pIpC) led to a 2.5-fold expansion of TP53-mutant cells in WT:TP53R172H/+ chimeras, whereas WT cells were depleted. This indicates that pro-inflammatory cues promote fitness advantage of TP53-mutant cells whilst suppressing antecedent clones. In summary, we present a comprehensive single-cell multi-omic analysis ofTP53-mediated transformation, providing unique insights into the evolution of chronic hematological malignancies towards an aggressive acute leukemia and of broader relevance to other cancer types.

Effect of RITA on TP53 Mutant Human Mantle Cell Lymphoma Cell Line and Its Mechanism

To investigate the effect of RITA on TP53 mutant human mantle cell lymphoma (MCL) cell line Mino and its possible mechanism. Mino cells were cultured in RPMI-1640 and treated with RITA at a concentration of 0-16 μmol/L for 24,48,72 hours. Cell viability was assessed by CCK-8 assay. The cells were treated by RITA (0-8 μmol/L) for 48 h, the cell apoptosis induced by RITA was detected by annexin V/PI flow cytometry. Western blot was performed to evaluate the expression of protein BCL-2, Caspase-3, Cleaved Caspase-3, PARP, MDM2, and P53 in Mino cells. After treatment with 0.5, 1, 2, 4, 8, and 16 μmol/L RITA for 48 h, the proliferation inhibition rate of Mino cells was (1.2±5.6)%, (14.9±4.9)%, (41.7±5.0)%, (61.8±2.4)%, (70.2±2.8)%, and (70.8±2.4)%, respectively. RITA could inhibit the proliferation of Mino cells significantly, and statistical analysis showed that the inhibition rate was increased with the increasing of RITA concentration (r=0.767). After the cells were treated by 4 μmol/L RITA for 24, 48, and 72 h, the proliferation inhibition rate was (25.2±3.8)%, (61.8±2.4)%, and (87.0±0.7)%, respectively. Satistical analysis showed that the inhibition rate was also increased with the increasing of treatment time (r=0.978). The apoptosis rate of Mino cells treated by 0, 2, 4, and 8 μmol/L RITA for 48 h was (5.4±0.4)%, (15.3±0.6)%, (38.7±1.7)%, and (50.8±1.1)%, respectively, and it showed dose-dependent manner (r=0.961). Western blot showed that with the increasing of RITA concentration, the BCL-2 protein expression was decreased in a dose-dependent manner (r=0.932), moreover, PARP cleavage and Caspase-3 activation were found, while the protein expression of MDM2 and P53 showed no change. RITA can inhibit the proliferation and induce the apoptosis of Mino cells significantly. The mechanism may be dependent on the Caspase pathway, but independent on the P53 pathway.

Co-Targeting Intrinsic and Extrinsic Apoptosis to Maximize Cell Death Induction in Venetoclax-Resistant AML Cells

Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations.We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments).We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p<0.01) was more effective, and APG2575 plus APG115 (180 d, p<0.01) most effectively extended survival compared to each drug alone. Triple combination treated mice lived longest (185 d), which was significantly longer than APG115 plus APG1387 and APG2575 plus APG1387 but did not reach statistical significance compared to APG2575 plus APG115. Data showed that triple and APG115+APG2575 combinations were most effective, followed by APG2575+APG1387, then APG115+APG1387, APG2575, or APG115, and finally APG1387.Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased.In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. DisclosuresCarter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

Targeting Polo-like Kinase 4 Triggers Polyploidy and Apoptotic Cell Death in TP53-Mutant Acute Myeloid Leukemia

Background: TP53 mutations in acute myeloid leukemia (AML) are associated with complex karyotype, high incidence of minimal residual disease (MRD), and high risk of relapse (Döhner et al., 2017; Giacomelli et al., 2018). While numerous novel treatment regimens, including the combination of the BCL2 inhibitor venetoclax (VEN) and hypomethylating agents (HMA), have emerged as partially effective treatments and resulted in higher remission rates in patients with TP53-mutant AML, full clearance of the mutant TP53 clone is rarely achieved and the majority of patients relapse (Short et al., 2021; Takahashi et al., 2016). The mechanisms responsible for response and relapse in TP53-mutant AML remain unclear and investigating novel mechanisms is critical to develop more effective therapies.Results: In order to shed light on the defective p53 signaling pathways underlying TP53 mutant AML, and to better understand mechanisms of resistance, we performed RNA-sequencing (RNA-seq) on FACS-sorted subpopulations using samples collected from TP53-mutant or TP53-wt high-risk AML patients. Samples were collected at diagnosis (DX) and post-treatment (POSTTX) (total number of samples n= 67, TP53-mutant=36, TP53-wt=31). Diagnostic samples include bulk AML, leukemic stem cells (LSCs), and post-treatment samples including bulk mononuclear cells (MNCS) and patient specific MRD (total n= 67, DX_Bulk=15, DX_LSCs=15, POSTTX_MNCs=14, POSTTX_MRD=23). Differential gene expression analysis of TP53-mutant samples indicates a positive enrichment of the following pathways: G2/M checkpoint, MYC targets, and mitotic spindle, among others. We focused here on genes associated with TP53-mutant AML enriched pathways, and identified a key regulator of centriole biogenesis, one of E2F targets: Polo-like kinase 4 (PLK4) as a potential target highly expressed in TP53-mutant AML samples . Previous publications showed that PLK4 is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing (Fischer et al., 2014; Li et al., 2005). Here we show that TP53-mutant AML samples lack the p53-dependent PLK4 repression and have higher levels of PLK4 compared to TP53-wt AML.To test the rigor of this finding, we interrogated the Munich Leukemia Laboratory (MLL) data base and analyzed their clinically annotated (e.g. karyotype, survival, complete blood counts, previous treatments ... etc) RNA-seq dataset of 726 AML samples (TP53-mutant=72, TP53-wt=654). TP53-mutant AML samples consistently showed significant PLK4 upregulation (p= 0.0003). We analyzed PLK4 expression and its correlation with TP53 mutations in The Cancer Dependency Map project dataset (1375 cell lines in 35 different types of cancers) (p= 0.004). Furthermore, we found significantly higher PLK4 protein levels in TP53-mutant AML MOLM13 cell lines when compared with syngeneic TP53-wt AML MOLM13 cells. Experimentally, we found that PLK4 inhibition using 25nM CFI-400945 in TP53-mutant AML MOLM13 cell lines triggers polyploidy > 2-fold higher than in TP53-wt AML MOLM13 cell lines 72 hours post treatment (Fig.1A p< 0.0001). Finally, we show that polyploidy is not reversible after drug removal and results in significantly increased levels of apoptotic cell death in TP53-mutant AML MOLM13 cells (Fig.1B).Conclusion: Our data suggest that TP53-mutant AML expresses higher levels of PLK4 in comparison to TP53-wt AML, and targeting PLK4 triggers polyploidy and apoptotic cell death in TP53-mutant AML. A clinical trial is ongoing testing the efficacy of PLK4 inhibition (CFI-400945) in AML (Clinical Trial ID: NCT04730258, TWT-202). [Display omitted] DisclosuresIssa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Borthakur: Takeda: Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; Astex: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy. Konopleva: Ascentage: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Sanofi: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Andreeff: Senti-Bio: Consultancy; ONO Pharmaceuticals: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Breast Cancer Research Foundation: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Medicxi: Consultancy; Amgen: Research Funding; AstraZeneca: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Syndax: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

Combined Inhibition of Bcl-2 and Mcl-1 Circumvents Resistance of TP53 Deficient/Mutant AML to BH3 Mimetics

AML patients with TP53 mutations have extremely poor clinical outcomes. This is due primarily to limited responses to available therapies including the highly promising FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents (DiNardo CD et al., Blood 2020), which resulted in CR/CRi rates of 70-95% and good tolerability in elderly patients (DiNardo CD et al., Lancet Oncol 2018 and Blood 2019). Apoptosis is regulated by anti- and pro-apoptotic proteins. While p53 does not directly regulate anti-apoptotic Bcl-2 proteins that are resistance factors for VEN, p53 transcriptionally up-regulates pro-apoptotic Bcl-2 proteins. Reverse phase protein array analysis of samples from newly-diagnosed AML patients found that pro-apoptotic Bax was significantly decreased in patients with TP53 mutations (Carter BZ, ASH 2019), which, as expected, diminished the effectiveness of Bcl-2 inhibition. Thus, strategies to target additional anti-apoptotic proteins, or increase pro-apoptotic proteins, are needed to enhance the efficacy of Bcl-2 inhibition in these patients.We determined protein levels of Bcl-2 family members in isogeneic Molm13 cells with TP53-knockout (KO), or with various hotspot TP53 mutations including R175H, Y220C, M237I, R248Q, R273H, and R282W. We observed markedly decreased Bax expression, to a less degree Bak decrease, and variable alterations in other Bcl-2 proteins in these cells compared to TP53-wild-type (WT) controls. We treated the aforementioned cells with VEN or the Mcl-1 inhibitor AMG 176 and found that TP53-KO or mutant cells were more resistant to both VEN and AMG 176 compared to WT controls. However, the combination of two inhibitors was highly synergistic in both settings, controls (CI = 0.2) and TP53-KO and mutant cells (CI < 0.1). To demonstrate that the decreased sensitivity to BH3 mimetics was, at least in part, mediated through Bax reduction in the TP53-mutant cells, we treated Bax knockdown (KD) Molm13 cells with VEN, AMG 176, or both. The Bax KD cells were resistant to VEN and AMG 176, while the combination of the two agents synergistically induced cell death.To establish potential clinical relevance of co-targeting Bcl-2 and Mcl-1 in TP53-mutant AML, we co-cultured cells from various TP53-mutant AML patients (n = 8) with mesenchymal stromal cells and treated them with VEN, AMG 176, or both. The combination synergistically induced cell death in both CD45 + leukemia blasts (CI values between 0.04 ± 0.04 to 0.34 ± 0.10) and CD34 + AML stem/progenitor cells (CI values between 0.07 ± 0.08 to 0.28 ± 0.14). RNA-sequencing of mononuclear and MRD cells of clinical samples (Issa G, ASH 2019) collected after induction therapy revealed that Mcl-1 expression was significantly higher in the TP53-mutated mononuclear and MRD cells compared to their WT counterparts (Fig. 1), which suggests that Mcl-1 contributes to treatment resistance and disease relapse. This further suggests that Mcl-1 inhibition should be incorporated in AML treatment, including VEN-based therapies, for patients with TP53 mutations.Finally, we treated NSG mice inoculated with isogeneic TP53-WT luciferase/GFP-labeled Molm13 and BFP-labeled TP53 R248W/R213* Molm13 cells (10:1) with VEN, AMG 176, or their combination. Only the combination treatment markedly decreased the number of GFP- and BFP-labeled cells in circulation and significantly prolonged mouse survival (median 23 d, 25 d, 24.5 d for control, VEN, AMG 176, respectively; and 45 d for VEN + AMG 176: P = 0.0007, 0.0009, and 0.0011 of combination vs. control, VEN, and AMG 176, respectively) (Fig. 2).Collectively, we demonstrate that decreased Bax contributes to resistance of TP53-mutant AML to BH3 mimetics. Mcl-1 expression positively impacts therapy resistance and disease reoccurrence in TP53-mutant AML. Thus, targeting Bcl-2 or Mcl-1 individually is insufficient and inhibition of both proteins is needed to shift cell fate from survival to death and circumvents resistance of TP53 deficient/mutant AML and AML stem/progenitor cells to BH3 mimetics. The concept warrants further clinical evaluation. [Display omitted] DisclosuresCarter: Syndax: Research Funding; Ascentage: Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Andreeff: Medicxi: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Syndax: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; Glycomimetics: Consultancy; Oxford Biomedica UK: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

TP53 Null AML Line Does Not Selectively Expand Under Lenalidomide Treatment

Lenalidomide is an immunomodulatory therapeutic (IMiD) with known anti-proliferative and anti-angiogenic effects, and is an approved therapy for both 5q- Myelodysplastic Syndromes and Multiple Myeloma (MM). This thalidomide analog is known to retarget the E3 ubiquitin ligase cereblon to differential ubiquitination, and therefore degradation, of specific substrates. While lenalidomide maintenance is known to promote progression-free survival, increased rates of therapy-related myeloid neoplasms (TMNs) have been observed in MM patients on IMiD maintenance therapy following autologous stem cell transplant. Of note, TP53 is the most commonly mutated gene in TMN arising after lenalidomide therapy (Mouhieddine et al. 2020). TP53 is also recurrently mutated in TMNs following chemotherapy, suggesting that genotoxic stress results in the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations in TP53 (TN Wong et al. 2018). Based on these observations, we hypothesize that lenalidomide may contribute to the development of TMN by providing a fitness advantage to HSPCs carrying TP53 mutations.To test this hypothesis, we generated TP53 -/- deficient MOLM13 AML cells using CRISPR-Cas9 gene editing. We confirmed guide editing efficiency by next generation sequencing of a bulk edited pool of cells and generated isogenic clones via outgrowth of singly sorted cells, in an accepted approach for the field (Boettcher et al. 2019). We validated clones via sequencing and western blotting, and identified no differences in basal proliferation characteristics. Next, we further compared TP53 -/- deficient clones' with TP53 +/+ clones' response to cisplatin treatment (0.05-10µM) and observed a 2.8 fold increase in IC50 for TP53 -/- deficient clones. Satisfied that TP53 -/- isogenic clones successfully recapitulated this known resistance phenotype, we examined the response to lenalidomide by mixing TP53 -/- and TP53 +/+(AAVS1 edited control) clones and measuring expansion under lenalidomide treatment. However, we observed substantial inter-clone heterogeneity, with widely varying cellular proliferation characteristics following clone mixing, despite our prior validation. Therefore, we shifted to using bulk edited MOLM-13 cells in order to mitigate the potential impact of off-target effects and strong selective pressure of singly sorted clones. We edited a pool of cells at either TP53 or the safe harbor locus AAVS1 , allowed cells to recover, and mixed the two edited populations before treating with PBS, vehicle (DMSO), 1µm or 10µM lenalidomide for up to 28 days. We sequenced both AAVS1 and TP53 loci to measure cellular expansion by tracking the fraction of unedited, safe harbor edited, and TP53-/- genomic DNA at 0, 3, 7, and 28 days. Contrary to our hypothesis, TP53 -/- cells did not expand over time in response to lenalidomide treatment, and we did not detect a difference in TP53 -/- cell expansion with lenalidomide treatment compared to vehicle control (Figure 1). While we did not detect a cell intrinsic effect of TP53 mutations on lenalidomide response, it is possible that there are cell extrinsic effects, such as alterations in the bone marrow microenvironment, that contribute to the development of TP53-mutant TMN. It is also possible that a more prolonged exposure of lenalidomide is needed to see a significant effect of TP53-mutant cell expansion. This is relevant, since patients may be maintained on lenalidomide therapy for years. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P24.13 Circulating Tumor Cells Predict Prognosis Following First-Generation EGFR-TKI Treatment in EGFR- and TP53-Mutant Non-Small Cell Lung Cancer

For advanced non-small cell lung cancer (NSCLC) with EGFR mutations, first-generation EGFR-TKIs have become the first-line standard treatment. Co-mutation of EGFR and TP53 may be indicated EGFR-TKI resistance, shorter PFS and OS. This study isolates and quantifies circulating tumor cells (CTCs) and evaluates patient prognosis before and after first-line treatment with EGFR-TKIs in advanced NSCLC with EGFR and TP53 mutation. Patients with advanced NSCLC with EGFR and TP53 mutation were treated with a first-generation EGFR-TKI using a standard daily dose. Continuous blood samples were collected at baseline (CTCs-d0) and 28 days (CTCs-d28), and the isolation by CTCBIOPSY® was used to detect CTCs. The CTCs results were divided into favorable (< 5 CTCs) and unfavorable (≥ 5 CTCs) groups. The median progression-free survival (PFS) of patients in the favorable group was significantly longer at baseline and after 28 days of treatment compared to those in the unfavorable group (p = 0.0055; p = 0.0003). After treatment, the PFS of patients with reduced CTCs was significantly better than those with no significant change in CTCs (p = 0.014). Patients with CTCs-d0 ≥ 5 and CTCs-d28 ≥ 5 had significantly lower PFS when compared to those with CTCs-d0 < 5 and CTCs-d28 < 5, respectively. This study confirmed for the first time that CTC count is closely correlated with prognosis in EGFR- and TP53-mutant advanced NSCLC following first-line treatment with first-generation EGFR-TKIs.

Suppression of autophagy promotes fibroblast activation in p53-deficient colorectal cancer cells

Deficiency of p53 in cancer cells activates the transformation of normal tissue fibroblasts into carcinoma-associated fibroblasts; this promotes tumor progression through a variety of mechanisms in the tumor microenvironment. The role of autophagy in carcinoma-associated fibroblasts in tumor progression has not been elucidated. We aimed to clarify the significance of autophagy in fibroblasts, focusing on the TP53 status in co-cultured human colorectal cancer cell lines (TP53-wild-type colon cancer, HCT116; TP53-mutant colon cancer, HT29; fibroblast, CCD-18Co) in vitro. Autophagy in fibroblasts was significantly suppressed in association with ACTA2, CXCL12, TGFβ1, VEGFA, FGF2, and PDGFRA mRNA levels, when co-cultured with p53-deficient HCT116sh p53 cells. Exosomes isolated from the culture media of HCT116sh p53 cells significantly suppressed autophagy in fibroblasts via inhibition of ATG2B. Exosomes derived from TP53-mutant HT29 cells also suppressed autophagy in fibroblasts. miR-4534, extracted from the exosomes of HCT116sh p53 cells, suppressed ATG2B in fibroblasts. In conclusion, a loss of p53 function in colon cancer cells promotes the activation of surrounding fibroblasts through the suppression of autophagy. Exosomal miRNAs derived from cancer cells may play a pivotal role in the suppression of autophagy.

Epigenetic Therapies in Ovarian Cancer Alter Repetitive Element Expression in a TP53-Dependent Manner.

Epithelial ovarian carcinomas are particularly deadly due to intratumoral heterogeneity, resistance to standard-of-care therapies, and poor response to alternative treatments such as immunotherapy. Targeting the ovarian carcinoma epigenome with DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi) increases immune signaling and recruits CD8+ T cells and natural killer cells to fight ovarian carcinoma in murine models. This increased immune activity is caused by increased transcription of repetitive elements (RE) that form double-stranded RNA (dsRNA) and trigger an IFN response. To understand which REs are affected by epigenetic therapies in ovarian carcinoma, we assessed the effect of DNMTi and HDACi on ovarian carcinoma cell lines and patient samples. Subfamily-level (TEtranscripts) and individual locus-level (Telescope) analysis of REs showed that DNMTi treatment upregulated more REs than HDACi treatment. Upregulated REs were predominantly LTR and SINE subfamilies, and SINEs exhibited the greatest loss of DNA methylation upon DNMTi treatment. Cell lines with TP53 mutations exhibited significantly fewer upregulated REs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the TP53-mutant cell line had significantly higher baseline expression of REs but upregulated fewer upon epigenetic treatment. In addition, p53 activation increased expression of REs in wild-type but not mutant cell lines. These data give a comprehensive, genome-wide picture of RE chromatin and transcription-related changes in ovarian carcinoma after epigenetic treatment and implicate p53 in RE transcriptional regulation. SIGNIFICANCE: This study identifies the repetitive element targets of epigenetic therapies in ovarian carcinoma and indicates a role for p53 in this process.