TP53-WT Cell Research Articles

The impact of TP53 mutations and use of the TP53-mutation-reactivating agent APR-246 on metastatic castrate-sensitive prostate cancer.

e17077 Background: TP53 mutations appear to be enriched over the spectrum of metastatic castration-sensitive prostate cancer (mCSPC) and are associated with worse survival outcomes. We chose to further explore the impact of dominant negative (DN) TP53 mutations on mCSPC progression and pro-metastatic behaviors in addition to studying the ability of APR-246, a small molecule targeting TP53 mutants, to blunt pro-metastatic behaviors. Methods: We retrospectively analyzed 531 mCSPC patients who underwent next generation sequencing. Patients were stratified by metastasis timing (synchronous if metastasis present at diagnosis or metachronous if arising after definitive treatment of localized disease) and the number of metastatic lesions (oligometastatic <5 or polymetastatic > 5 lesions). Tumors were classified based on TP53 mutation status (missense, truncating, or wild-type [WT]) and dominant negativity, which was defined as production of a mutant protein that reduces the residual WT protein's transcriptional activity according to World Health Organization TP53 database. Clinical outcomes were radiographic progression-free survival (rPFS) and overall survival (OS), evaluated with Kaplan-Meier and multivariable Cox regression. To verify the impact of TP53 mutation on metastasis, we created isogenic 22Rv1 prostate cancer cell lines that carried either TP53 WT or TP53 R175H and tested this mutation for migration, invasion, and anchorage-independent growth. APR-246 (25-80 µM) was tested for anti-metastatic properties in vitro and anti-tumor growth in 22Rv1 xenografted nude mice. Results: In our cohort, 155 (29.2%) had a TP53 mutation, which mostly occurred in the DNA-binding domain (85.16%). DN TP53 mutations were associated with more aggressive disease states: DN TP53 mutations were enriched in patients with synchronous (vs. metachronous: 20.7% vs. 6.3%, p < 0.01) and polymetastatic disease (vs. oligometastatic: 14.4% vs. 7.9%, p < 0.01). On multivariable analysis, DN TP53 mutations were correlated with shorter rPFS (HR = 1.97, 95%CI: 1.31-2.98, p < 0.01) and OS (HR = 2.05, 95%CI: 1.14-3.68, p = 0.02) compared to those with TP53 WT. In vitro, 22Rv1 cells with DN TP53 R175H mutations had increased ability to migrate, invade, and form colonies compared to TP53 WT cells. APR-246 treatment of TP53 R175H mutants blunted the pro-metastatic effects of the cell line in vitro (p < 0.01 for all assays by unpaired t-test). Interestingly, APR-246 inhibited xenograft tumor growth of 22Rv1 TP53 R175H prostate cancer cells (p < 0.0001 by two-way ANOVA). Conclusions: DN TP53 mutations were associated with poorer survival outcomes for mCSPC patients. DN TP53 mutations also promoted prostate cancer pro-metastatic behaviors in vitro, which was effectively counteracted by APR-246, making it a promising treatment option that should be explored further in early-phase clinical studies.

Combined inhibition of BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance of TP53-mutant acute myeloid leukemia to individual BH3 mimetics

TP53-mutant acute myeloid leukemia (AML) respond poorly to currently available treatments, including venetoclax-based drug combinations and pose a major therapeutic challenge. Analyses of RNA sequencing and reverse phase protein array datasets revealed significantly lower BAX RNA and protein levels in TP53-mutant compared to TP53–wild-type (WT) AML, a finding confirmed in isogenic CRISPR-generated TP53-knockout and -mutant AML. The response to either BCL-2 (venetoclax) or MCL-1 (AMG176) inhibition was BAX-dependent and much reduced in TP53-mutant compared to TP53-WT cells, while the combination of two BH3 mimetics effectively activated BAX, circumventing survival mechanisms in cells treated with either BH3 mimetic, and synergistically induced cell death in TP53-mutant AML and stem/progenitor cells. The BH3 mimetic–driven stress response and cell death patterns after dual inhibition were largely independent of TP53 status and affected by apoptosis induction. Co-targeting, but not individual targeting of BCL-2 and MCL-1 in mice xenografted with TP53-WT and TP53-R248W Molm13 cells suppressed both TP53-WT and TP53-mutant cell growth and significantly prolonged survival. Our results demonstrate that co-targeting BCL-2 and MCL-1 overcomes BAX deficiency-mediated resistance to individual BH3 mimetics in TP53-mutant cells, thus shifting cell fate from survival to death in TP53-deficient and -mutant AML. This concept warrants clinical evaluation.

Splicing Modulation Results in Aberrant Isoforms and Protein Products of p53 Pathway Genes and the Sensitization of B Cells to Non-Genotoxic MDM2 Inhibition.

Several molecular subtypes of cancer are highly dependent on splicing for cell survival. There is a general interest in the therapeutic targeting of splicing by small molecules. E7107, a first-in-class spliceosome inhibitor, showed strong growth inhibitory activities against a large variety of human cancer xenografts. Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous hematologic malignancy, with approximately 90% of cases being TP53 wild-type at diagnosis. An increasing number of studies are evaluating alternative targeted agents in CLL, including MDM2-p53 binding antagonists. In this study, we report the effect of splicing modulation on key proteins in the p53 signalling pathway, an important cell death pathway in B cells. Splicing modulation by E7107 treatment reduced full-length MDM2 production due to exon skipping, generating a consequent reciprocal p53 increase in TP53WT cells. It was especially noteworthy that a novel p21WAF1 isoform with compromised cyclin-dependent kinase inhibitory activity was produced due to intron retention. E7107 synergized with the MDM2 inhibitor RG7388, via dual MDM2 inhibition; by E7107 at the transcript level and by RG7388 at the protein level, producing greater p53 stabilisation and apoptosis. This study provides evidence for a synergistic MDM2 and spliceosome inhibitor combination as a novel approach to treat CLL and potentially other haematological malignancies.

Epichaperome Inhibition Targets Kinase and TP53 Mutant Therapy Resistant AML

The BCL-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, improves outcomes for many patients with AML and is now considered standard of care for patients older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months; 2.5 months after relapse. Mutations of FLT3, PTPN11, and RAS, leading to constitutive activation of cell survival signaling, and mutations of TP53 have been identified as key determinants of low response rates, early relapse and death. Mutant TP53 cells are also characterized by activation of signaling kinases. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. Epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, epichaperome inhibition has the potential of eliminating resistant subclones before they become dominant and cause relapse. In contrast to other HSP90 purine analog inhibitors, PU-H71(zelavespib) is a competitive inhibitor specific for the conformationally changed ATP binding site of HSP90 when incorporated into epichaperomes. We here investigate the therapeutic potential of targeting epichaperomes with PU-H71 in TP53 and oncogenic kinase mutant AML. Western blot analysis showed increased HSP90 and several signaling proteins such as STAT3 and c-Myc in TP53-knockout and mutant Molm13 cells generated by CRISPR/cas-9, compared to isogeneic wild-type (WT) controls. Using fluorochrome-labelled PU-H71 and flow cytometry, we demonstrated the presence of epichaperome in AML and AML stem/progenitor cells with TP53 and/or RAS mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively killed AML cells, including those with acquired resistance to BH3 mimetics and those with various FLT3 mutants. PU-H71 targets AML cells and stem/progenitor cells with TP53 mutations, and prolonged survival of TP53-mutant AML xenograft mice with minimal effects on mouse bone marrow LSK cells and hematopoiesis. PU-H71 increased the expression of pro-apoptotic BIM and the efficacy of VEN in AML cells and AML stem/progenitor cells with TP53 and/or RAS mutations (combination index < 1). Importantly, in a 1,000: 1 mixture of TP53-WT and TP53-R248W mutant isogenic Molm13 cells, MDM2 inhibitor nutlin3a selectively killed TP53-WT but enriched TP53-mutant Molm13 cells, as expected, and VEN treatment also favored the outgrowth of TP53-mutant cells. However, PU-H71 effectively killed TP53-WT and mutant cells (Fig. 1): 10 million TP53-WT cells were reduced to 335 cells, and 10,000 TP53-mutant cells to 26. The activity of PU-H71 against both TP53-WT and mutant AML cells was further enhanced by VEN in vivo in a xenograft model of mixed (10:1) TP53-WT and mutant Molm13 cells. Our data support the concept that the epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring TP53 and/or oncogenic kinase mutations. Inhibition of epichaperomes by PU-H71 targets AML cells/stem/progenitor cells, enhances VEN activity, and prevents the outgrowth of nutlin- or VEN-resistant TP53-mutant AML cells. This concept has entered clinical evaluation in MPN-derived secondary AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Deacetylase Inhibition Restores Therapy Response in TP53mutated Acute Lymphoblastic Leukemia to Chemotherapy

Background: Although TP53 is the most frequently mutated gene in cancer, in Acute Lymphoblastic leukemia (ALL), mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse TP53 aberrations are found in about 12% of the patients and predict a very poor outcome. As a consequence, relapsed TP53 deleted ALL is now classified as 'very high risk, together with t(1;19) and t(17;19) ALL. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance not only to classical chemotherapeutics, but also newly introduced immunotherapies such as bi-specific T cell engagers such as Blinatumomab or CAR-T cell therapies. Therefore, there is an urgent clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods: Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wildtype and knockout models in the TP53WT B-cell precursor ALL (BCP-ALL) cell lines Nalm6 and RCH-ACV. After confirming the presence of TP53 deletions, we treated these isogenic models with a panel of treatment protocol drugs, demonstrating that TP53 loss negatively affects response to therapy to most contemporary drugs used in the treatment of relapsed ALL. To identify drug combinations that may reverse TP53 mediated therapy resistance, we assessed the viability of these isogenic models in a drug screen containing 198 (pre)clinical drugs. The most interesting targets were subsequently combined with the previously tested treatment protocol drugs to determine potential synergies. Effects on cell viability were determined by amine exposure and MTT assay and confirmed by measuring PARP cleavage. Additionally, positive drug interactions were validated in vivo using immunocompromised mice engrafted with RCH-ACV p53KO cells expressing a luciferase reporter gene. Results: We observed that loss of p53 protects BCP-ALL cell lines Nalm6 and RCHACV from drug-induced apoptosis, leading to increased resistance to all drugs used currently in the treatment of relapsed ALL, except for Asparaginase. In a subsequent drug screen, we found that the histone deacetylase (HDAC) 1/2 inhibitor romidepsin shows strong single agent activity at single nanomolar concentrations. Moreover, romidepsin was found to strongly synergize with contemporary ALL treatment drugs in TP53KO and TP53WT cells (Figure 1A). This effect was strongest in TP53KO cells, effectively reversing therapy resistance induced by TP53 loss. Gene expression profiling suggest that romidepsin may lead to reactivation of TP53 target genes in response to cellular stress, even in the absence of functional p53. Finally, preliminary mouse experiments using TP53 deleted RCH-ACV cells showed that romidepsin improves the response to cytarabine in TP53 aberrant leukemia in vivo (Figure 1B) without signs of added toxicity. A mouse trial testing a potential synergy between romidepsin and cytarabine using TP53 deleted patient derived xenografts (PDX) is currently underway. Conclusions: Loss of p53 protects BCP-ALL cells from currently used (chemo)therapy strategies. Romidepsin can restore response to therapy in TP53 deleted ALL, presumably by reactivation of p53 target genes in response to cellular stress. Therefore, romidepsin may be used to improve therapy response in relapsed TP53 deleted ALL. Since romidepsin already has FDA approval for use in other hematological malignancies, our findings may be rapidly translated into clinical practice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TP53 CRISPR/Cas9 Reveals Strict BAX Dependence in the Response to BH3 Mimetic Targeting MCL1

Background In multiple myeloma (MM), 1q gain/amplification (1q+) and deletion of the short arm of chromosome 17 (del17p), encompassing MCL1 and TP53, respectively, as well as the t(4;14) translocation are adverse events that reduce both event-free survival and overall survival of patients. The genome gatekeeper p53 is well known for its multiple roles in the regulation of mitochondrial apoptosis: p53 induces early direct transactivation of the BH3-only protein PUMA (BBC3) and the death effector BAX (BAX) and late direct-to-indirect transactivation of NOXA (PMAIP1) and BAK (BAK1). To investigate the direct impact of loss of p53 function in myeloma cells, we generated TP53 CRISPR/Cas9 isogenic cells from cell lines harboring t(4;14) and 1q+. Materials and Methods To knockout the expression of all p53 isoforms in two TP53wt t(4;14)+ 1q+ myeloma cell lines, NCI-H929 and XG7 cells were sequentially transduced with 2 lentiviruses expressing Cas9 and a doxycycline-inducible TP53-specific sgRNA targeting exon 5. TP53-/-clones (n=3) and control clones (n=3) were derived for each cell line by limiting dilution. Results RNAseq analysis was performed in triplicate in the 12 clones of the two MM cell lines: analysis of the 6 TP53-/-clones versus the 6 control clones showed significant changes (log2FC>0.4, p>0.05) in the expression of 92 genes under steady-state conditions (and of more than 3000 upon nutlin3a-induced p53 accumulation) : 27% of the genes were known to be direct p53 target genes, 39% were known to be indirectly regulated, and 35% of the genes were not identified as p53 targets in solid cancer cell lines (Andrysik Z Genome Res 2017). Among the 51 downregulated genes, only 2 belonged to apoptosis pathways, namely BAX and TNFRSF10B, both direct early targets of p53. Of note, the expression of BAK1, PMAIP1 or BBC3 was not significantly modified. To directly assess the impact of BAX decrease in the 6 TP53-/- clones, we used 3 BH3 mimetics specific for BCL2, Venetoclax, MCL1, S63845, and BCLXL, A1155463. The XG7 and NCI-H929 cell lines were predominantly resistant to Venetoclax and to A1155463, and TP53 knockout did not alter responses. In contrast, as compared to control clones, the response to S63845 was decreased 5.7 to 7.4-fold in NCI-H929 TP53-/- (LD50 median values increased from 7 to 40nM, p<0.0001) and XG7 TP53-/- clones (LD50 median values increased from 25 to 186nM, p=0.0007), respectively. This result suggested a major role of BAX in the response to S63845. Indeed, BAX silencing significantly inhibited S63845-induced cell death while BAK silencing had no protective effect, demonstrating the BAX dependence of the S63845 response. Of note, this strong dependence on p53-dependent BAX expression was not limited to XG7 or NCI-H929 : in 21 TP53abnormal myeloma cell lines, BAX expression (GEP) was 1.7-fold lower than in 11 TP53wt cell lines (LD50 median values 34 vs. 59, p=0.0004), and sensitivity to S63845 was also significantly lower in TP53abn MM cell lines compared to MM TP53wt (LD50 median values 75 nM vs. 12 nM, p=0.0296). The resistance of TP53-/- cells to S63845 was overcome by the addition of Venetoclax, although the death remained lower in TP53-/- clones compared to control clones (p=0.001). BAX and BAK activation and MCL1 co-immunoprecipitation assays further demonstrated the central role of MCL1/BAX expression and complexes in the response to S63845. In 63 consecutive independent samples from patients with MM (23 at diagnosis, 40 at relapse), 33 1q+ and 30 1q-, we show that 1q gain/amplification favored response to 25 nM S63845 alone (death median 16% in 29 1q- vs 30% in 34 1q+, p=0.0132) and had a trend in combination with 300 nM Venetoclax (80% vs 89%, p=0.152). Finally, we show that 1q was sufficient to induce responses to S63845 plus Venetoclax regardless of del17p status (death median 30% in 26 1q+ del17p- vs. 40% in 6 1q+ del17p+ samples). To analyze the response to the combination of S63845 and Venetoclax at the single cell level, we performed scRNASeq analysis in t(4;14)+ 1q+ del17p+/- patients and determined the transcriptomic profile of cells surviving the combination: we show that BH3 mimetics combination heterogeneously induced death in and between patient's samples and we identified a resistance signature that is under evaluation. All these results support the S63845 / Venetoclax combination for patients with double (1q+, del17p) to triple (t(4;14), 1q+, del17p) hit. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antitumor effect of WEE1 blockade as monotherapy or in combination with cisplatin in urothelial cancer

Overcoming cisplatin (CDDP) resistance is a major issue in urothelial cancer (UC), in which CDDP‐based chemotherapy is the first‐line treatment. WEE1, a G2/M checkpoint kinase, confers chemoresistance in response to genotoxic agents. However, the efficacy of WEE1 blockade in UC has not been reported. MK‐1775, a WEE1 inhibitor also known as AZD‐1775, blocked proliferation of UC cell lines in a dose‐dependent manner irrespective of TP53 status. MK‐1775 synergized with CDDP to block proliferation, inducing apoptosis and mitotic catastrophe in TP53‐mutant UC cells but not in TP53‐WT cells. Knocking down TP53 in TP53‐WT cells induced synergism of MK‐1775 and CDDP. In UMUC3 cell xenografts and two patient‐derived xenograft lines with MDM2 overexpression, in which the p53/cell cycle pathway was inactivated, AZD‐1775 combined with CDDP suppressed tumor growth inducing both M‐phase entry and apoptosis, whereas AZD‐1775 alone was as effective as the combination in RT4 cell xenografts. Drug susceptibility assay using an ex vivo cancer tissue‐originated spheroid system showed correlations with the in vivo efficacy of AZD‐1775 alone or combined with CDDP. We determined the feasibility of the drug susceptibility assay using spheroids established from UC surgical specimens obtained by transurethral resection. In conclusion, WEE1 is a promising therapeutic target in the treatment of UC, and a highly specific small molecule inhibitor is currently in early phase clinical trials for cancer. Differential antitumor efficacy of WEE1 blockade alone or combined with CDDP could exist according to p53/cell cycle pathway activity, which might be predictable using an ex vivo 3D primary culture system.

Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a.

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.

Cell-cycle arrest and senescence in TP53-wild type renal carcinoma by enhancer RNA-P53-bound enhancer regions 2 (p53BER2) in a p53-dependent pathway

TP53 is a classic tumor suppressor, but its role in kidney cancer remains unclear. In our study, we tried to explain the role of p53 in kidney cancer through the p53-related enhancer RNA pathway. Functional experiments were used to explore whether P53-bound enhancer regions 2 (p53BER2) has a role in the cell cycle and senescence response of TP53-wild type (WT) renal cancer cells in vitro or vivo. RNA-sequencing was used to identify the potential target of p53BER2. The results showed that the expression level of P53BER2 was downregulated in renal cancer tissues and cell lines, further dual-luciferase experiments and APR-256-reactivated experiments showed p53BER2 expresses in a p53-dependent way. Moreover, knockdown p53BER2 could reverse nutlin-3-induced cytotoxic effect in TP53-WT cell lines. Further exploration showed the downregulation of p53BER2 could reverse nutlin-3-induced G1-arrest and senescence in TP53-WT cell lines. What is more, the knockdown of p53BER2 showed resistance to nutlin-3 treatment in vivo. Additionally, we found BRCA2 could be regulated by p53BER2 in vitro and vivo; further experiment showed p53BER2 could induce cell-cycle arrest and DNA repair by mediating BRCA2. In summary, the p53-associated enhancer RNA-p53BER2 mediates the cell cycle and senescence of p53 in TP53-WT renal cancer cells.

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Abstract NG02: Genomic evolution of cancer models: Perils and opportunities

Low-frequency TP53 hotspot mutation contributes to chemoresistance through clonal expansion in acute myeloid leukemia.

TP53 mutations (TP53mut) in AML patients associate with poor prognosis that may affect therapy and outcome. In addition to TP53 mut patients, TCGA AML patient sequencing data shows that there are around 3% of patients have detectable low frequency TP53mut reads. Importantly, these patients showed worse outcome as compared to the TP53 wild type (TP53wt) patients. We have studied the effect of low frequency TP53mut in two AML cell lines, OCI-AML2 and MV4–11. Both cells have low frequency single hotspot TP53mut. Interestingly, the resistant cells derived from both lines have homogeneous TP53mut. TP53mut clones isolated from the parental cells also show increased chemoresistance potential and have higher population of leukemia stem cell (LSC) maker positive cells, a characteristic of chemoresistant cells. When mixed with TP53wt cells, the TP53mut cells show survival advantage suggesting its potential to develop chemoresistance. We previously showed that histone deacetylase inhibitor Romidepsin can re-sensitize chemoresistant cells by eradicating LSC marker positive cells. Here we further show that Romidepsin can reactivate p53 targeted genes which are dysregulated in TP53mut cells and preferentially targets TP53mut subpopulation. Therefore, our study shows that low frequency TP53mut is linked to chemoresistance and shed lights on therapeutic strategies for treatments on chemoresistance.

Targeting Aurora Kinase B with AZD2811 Enhances Venetoclax Activity in TP53-Mutant AML

Background: Recent data suggests that older patients with acute myeloid leukemia (AML) have a higher frequency of poor risk TP53 mutations (TP53mut) than previously suspected. Patients with TP53mut have a very poor prognosis, making this AML sub-group an area of high unmet therapeutic need. Although recent clinical trials suggest promising response rates for the BCL-2 inhibitor venetoclax (Ven) in combination with DNA methyltransferase inhibitors (DNMTi) or low-dose cytarabine, survival outcomes remain poor among patients with TP53mut (Strickland et al, EHA 2018). Methods and Results: To identify novel therapies effective against TP53mut AML, isogenic TP53 knockout cells (TP53 KO) were generated by CRISPR/Cas9 in OCI-AML3, MV4;11 and MOLM-13 human AML cell lines. In a drug screen of 50 compounds, we identified AZD2811, an aurora kinase B inhibitor, which potently killed AML cells independently of TP53 genotype (Figure 1). We hypothesised that AZD2811 would synergise with Ven to overcome venetoclax-resistance in TP53-mutant AML. Consistent with this, AZD2811 and venetoclax showed strong synergy (Loewe score &lt;4) in 6 AML cell lines independent of TP53 genotype, and combined treatment displayed highly efficacious activity in both TP53 KO cells and wild type control cells in vitro (Figure 2). Immunoblot of treated cells revealed that AZD2811 depleted MCL-1 expression in the majority of cases, a known cause of venetoclax resistance. AZD2811 was also active in killing Bax/Bak knockout cells, suggesting that the mechanism of AZD2811 may not be limited to the intrinsic apoptosis pathway. Using a competitive growth assay incorporating a fluorescent reporter for TP53 KO cells, we showed the relative resistance of TP53 defective cells to Ven and decitabine (DEC) alone, as well as Ven + DEC, compared to wild type cells. In contrast, TP53 KO cells showed no competitive growth advantage when exposed to an initially sub-lethal dose of AZD2811 (10 nM), compared to TP53 WT cells. Interestingly, continuous exposure to AZD2811 resulted in a catastrophic collapse in cell viability on day 10, unlike comparator anti-leukemic agents (Figure 4). In vivo efficacy for nanoparticle formulated AZD2811 (NP) was observed in SCID mice xenografted with HL-60 cells (known to be TP53 mutant), where combined treatment of AZD2811NP and Ven significantly prolonged animal survival (Figure 3). Remarkably, combined AZD2811NP and Ven treatment was more effective than Ven + azacitidine treatment in vivo. Patient-derived xenograft (PDX) models to verify the activity of AZD2811NP + venetoclax against primary AML cases with TP53 mutations will be presented at the meeting. Conclusions: We report for the first time that AZD2811NP can overcome venetoclax resistance in TP53-mutant AML in vitro and in vivo. These findings therefore, support the clinical investigation of combined aurora kinase and BCL-2 targeting in the clinic for patients with TP53-mutant AML, who currently lack effective treatment options. Disclosures Urosevic: AstraZeneca: Employment. Polanska:AstraZeneca: Employment. Cosaert:AstraZeneca: Employment. Pease:AstraZeneca: Employment, Equity Ownership. Travers:AstraZeneca: Employment. Wei:Pfizer: Honoraria; Janssen: Honoraria; Walter and Eliza Hall Institute: Other: former employee, Patents &amp; Royalties: receives a fraction of its royalty stream related to venetoclax; AbbVie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Macrogenics: Honoraria; Astellas: former employee, Honoraria; Genentech: Honoraria; Servier: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

CRISPR-Cas9 Screen Identifies XPO7 As a Potential Therapeutic Target for TP53-Mutated AML

Acute myeloid leukemia (AML) is a devastating disease with a long-term survival rate of less than 30%. While AML is a genetically heterogeneous disease, TP53 mutation is among the most powerful risk factors in AML, underscoring the critical need to devise a novel therapeutic strategy for TP53-mutated AML. To identify genes/pathways whose loss are 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 are vulnerable to TP53 deficiency. Importantly, sgRNAs targeting Trp53 were enriched only in Trp53-WT AML cells, but not in Trp53-KO cells. Furthermore, sgRNAs targeting Mdm2, which encodes a E3 ubiquitin-protein ligase for Trp53 protein, were depleted only in Trp53-WT cells, attesting to the validity of our experimental system. 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 (Yamauchi et al. Cancer Cell 2018), 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, either CRISPR/Cas9-mediated Xpo7 depletion or shRNA-mediated Xpo7 knockdown significantly delayed cell cycle progression and suppressed cell growth only in Trp53-KO AML cells, but not in WT cells. These findings were also validated using the DepMap (https://depmap.org), a publicly-available CRISPR/Cas9 dropout screen dataset. Significantly, XPO7 dependency was most correlated with that of TP53 and particularly evident in TP53-WT cell lines. 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, supporting the notion that Xpo7 could function as a tumor suppressor when Trp53 function is intact and evident. In a stark contrast, the same sgRNAs, which target coding exons, were mostly depleted in Trp53-KO AMLs. Importantly, sgRNAs targeting the Xpo7 UTRs were unchanged/neutral regardless of experimental conditions (e.g. Trp53 status, MDM2 inhibitor treatment), serving as valid control for the experiments. 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 were significantly decreased, while those in the cytoplasm were increased upon Xpo7 depletion, revealed by Western blot. These data suggest that Xpo7 retains WT-Trp53 in the nucleus and functions as a Trp53-dependent tumor suppressor in Trp53-WT AML. Finally, to explore functional significance of XPO7 in human AML, we assessed correlation between XPO7 expression levels and AML subtypes and/or genetic background using publicly-available datasets. XPO7 mRNA levels were significantly upregulated in TP53-mutated AMLs in the TCGA datasets (Ley et al. NEJM 2013). XPO7 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 knockdown significantly suppressed proliferation of HEL, a human AEL cell line harboring TP53 mutation. In summary, we identified a synthetic lethal relationship between TP53 and XPO7. Our study may facilitate the development of novel therapeutic strategies for TP53-mutated AML, such as AEL. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

CBMT-32. EGFR SUPPRESSES p53 FUNCTION THROUGH DNA-PKcs BINDING TO p53: NOVEL CROSSTALK BETWEEN EGFR AND TP53 IN GLIOBLASTOMA

Abstract Comprehensive analysis of the TCGA Glioblastoma database has identified 3 prognostically relevant subgroups (Proneural, Classical and Mesenchymal). Interestingly, it has also revealed a near mutual exclusivity of EGFR amplification and TP53 mutations. There are only a few tumors with both amplified EGFR and mutant TP53. We decided to investigate the functional relationship between EGFR and TP53 relative to tumorigenesis and tumor proliferation. We selected from our molecularly characterized GSC bank a series of EGFR amplified+TP53wt/TP53mut lines for this study. Here we report that EGFR amplification overrides the effect of p53wt but co-regulates tumorigenesis with TP53R175H mutation. We show that CRISPR EGFR knockout in TP53wt cells leads to complete loss of cell growth suggesting that EGFR is essential for cell survival, providing growth signal and suppression of p53wt function. We further show that EGFR induces physical binding between p53wt and DNA-PKcs, which suppresses p53wt by inhibiting p53 phosphorylation at S15. The knockdown of DNA-PKcs restores p53wt anti-tumor function. In contrast, GSC262 harboring TP53R175H mutation is the only GSC that survives after EGFR knockout, suggesting that this specific mutation has a gain of function (GOF) to sustain growth without EGFR. This is confirmed by dual knockout of EGFR and TP53R175H showing no surviving cells. Further investigation shows both EGFR and p53R175H induce tumor glycolysis, the major energy metabolism and heightened growth signal for this group. We further show that EGFR mediated suppression of p53R175H is through DNA-PKcs binding to p53R175H and reduced S15 phosphorylation. Taken together, this study reveals 1) EGFR amplification is essential for tumor growth in TP53wt GSCs; 2) novel crosstalk between EGFR and p53 through DNA-PKcs. 3) EGFR suppresses p53wt function by providing the energy required for tumor growth through inducing glycolysis, and 4) TP53R175H is a GOF mutation that sustains life without EGFR by inducing glycolysis.

IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression

BackgroundImmune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma.MethodsWe analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway.ResultsFor TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53L22Q,W23S, a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53L22Q,W23S in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression.ConclusionsWhile having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.

P53 regulates CD46 expression and measles virus infection in myeloma cells

AbstractIn this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53wt cell lines, but not the TP53abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.

Abstract 1872: Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors

Abstract Introduction: The wild-type (wt) p53-inducible protein phosphatase WIP1 (gene PPM1D), is frequently mutated in solid tumors and in blood of healthy-individuals, but its role is still unknown in Acute Myeloid Leukemia (AML). WIP1 suppresses p53 after cell-stress. The aim of this study is to investigate whether the inhibition of WIP1 (GSK2830371) could increase the sensitivity to MDM2 inhibitors (Nutlin-3a) in order to obtain a novel therapeutic strategy for AML patients restoring p53 activity. Methods: Whole Exome Sequencing (WES) and targeted Next Generation Sequencing (NGS) have been performed to investigate the mutational state of PPM1D in newly diagnosed AML patients. We performed in vitro WST-1 cell-viability and Annexin V-PI apoptosis assays in AML cell lines (MOLM-13, MV-4-11) and AML primary cells. Results: WIP1i and Nut-3a reduced the cell viability as single agent in all the treated cells in a time and dosage-dependent manner. The IC50 values in TP53-wt cells showed that MOLM-13 (10 υM) are more sensitive than MV-4-11 (20 υM) to WIP1i. Combination index analyses confirmed that WIP1i and Nut-3a had a synergistic effect (C.I.&amp;lt;1) in both cell lines. Annexin V/PI staining performed after treatment with different concentration of Nut-3a (0.5 υM for MOLM-13, 15 υM for MV4-11) and WIP1 inhibitor (10 υM for MOLM-13, 20 υM for MV4-11) demonstrated that the combination significantly increased the number of apoptotic cells in comparison to single agents. In particular, after 48 hours of incubation with Nut-3a and WIP1i as single agents, the percentage of apoptotic cells was 34.4% and 21.4% for MOLM-13 and 17% and 18% for MV-4-11, respectively; while the combination induced 71.6% and 54.5% of apoptotic cells. The decrease of cells in G2/M phase induced by single agents, was enhanced by the drugs combination in both cell lines and, in particular, MV-4-11 showed an increasing percentage of S phase cells allowing us to hypothesize that the apoptotic death induced by the combination of drugs occurs when cells are blocked in S phase. AML primary cells were also tested, showing a similar pattern when the two drugs are combined. Finally, by WES and targeted-NGS, 7 mutations have been detected in 2/37 and 4/96 patients. One patient harbored a truncating mutation on exon 6, known to be gain of function, also confirmed by Western Blot (WB), increasing the inhibition of p53; while the other mutations affected the exon 1 and have not been described yet. Conclusions: This pre-clinical study allows us to affirm for the first time that WIP1i potentiates the sensitivity to Nut-3a in AML. Further studies on TP53-mutated and wt cells will help to confirm these preliminary data, Gene Expression Profile and WB will identify the proteins playing a relevant role in the biological alterations induced by WIP1i. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giorgia Simonetti, Andrea Ghelli Luserna di Rorà, Antonella Padella, Samantha Bruno, Cristina Papayannidis, Giovanni Marconi, Mariachiara Abbenante, Michele Cavo, Giovanni Martinelli. Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1872.

Abstract LB-210: Abrogation of mutant TP53 radiation resistance by lithium induced CTNNB1 (β-catenin) activation in medulloblastoma.

Abstract Introduction: Medulloblastoma (MB) is the most common paediatric malignant brain neoplasm. 30 % of patients will reccur and die of their tumour. TP53 mutations confer radiation resistance; we have recently demonstrated that somatic mutations in TP53 are predictive of poor survival in patients with MB. Patients with both TP53 and WNT activating CTNNB1 (β-catenin) mutations demonstrate improved survival compared to patients with TP53 mutations alone. WNT pathway is abrogated in 10% of MB. Lithium Chloride (LiCl) treatment constitutively activates WNT pathway by inhibition of Gsk3β-mediated degradation of β-catenin. Radiation therapy is key treatment in MB; hence we hypothesized that activation of WNT/β-catenin signalling can rescue radiation resistance of TP53 mutant phenotype, sensitize MB cells to radiation and can be achieved with administration of LiCl. Methods: Human MB, TP53 wild type (wt) and mutant (mut), all CTNNB1 wt cell lines were used to investigate radiation response and effect of LiCl on cell survival by clonogenic assays. Functional roles of TP53 and CTNNB1 mutations in response to LiCl/radiation treatments were assessed by transfection of mutant genes on a wild type background. Double strand (DS) DNA damage-repair was assessed by γH2AX immunofluorescence (IF). Normal neuronal stem cells were used to assess effects of LiCl/radiation on normal brain tissue by clonogenic assay. Results: TP53 mut MB cells were resistant to radiation compared to TP53 wt cells by clonogenic assay (p&amp;lt;0.01), accompanied by more efficient DNA damage response manifested by lower numbers of γH2AX foci. Transfection of R175H dominant-negative TP53 mutation to wt cells resulted in increased clonogenic potential post-radiation (p&amp;lt;0.01). MB cells transfected with CTNNB1 S33Y mutation revealed increased radiosenstivity especially in TP53 mut MB (p&amp;lt;0.01). LiCl treatment resulted in nuclear translocation of β-catenin and signalling activation by luciferase assay, which is consistent with activation of WNT pathway. Treatment with LiCl increased radiation sensitivity of both wt and mut TP53 cells (p&amp;lt;0.01; p&amp;lt;0.05 respectively), accompanied by increased DS DNA damage and delayed repair in both lines by γH2AX IF. LiCl treatment of normal neuronal stem cells did not sensitize them to radiation suggesting that LiCl is a tumour specific treatment. Conclusions: Constitutive activation of β-catenin by LiCl sensitizes TP53 mut MB cells to radiation possibly through activation of β-catenin/TCF mediated transcription and impaired DS DNA repair response. These findings may explain survival advantage in CTNNB1 mut MB patients harbouring TP53 mutation. Knowing safety of LiCl and its neuroprotective capacity in normal neuronal cells, oral administration of LiCl may represent an attractive novel therapy for MB patients. Investigations of the molecular mechanisms governing this phenomenon are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-210. doi:1538-7445.AM2012-LB-210