The stress-associated chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing oncoproteins. Most inhibitors target the key component, heat-shock protein 90 (HSP90). Although HSP90 inhibitors are highly tumor-selective, they fail in clinical trials. These failures are partly due to interference with a negative regulatory feedback loop in the heat-shock response (HSR): in response to HSP90 inhibition, there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock-factor 1 (HSF1). We recently identified that wild-type p53 reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here, we test whether in HSP90-based therapies, simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. We found that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids, and patient-derived organoids (PDOs). Mechanistically, upon combination therapy, CRC cells upregulate p53-associated pathways, apoptosis, and inflammatory pathways. Likewise, in a CRC mouse model, dual HSF1-HSP90 inhibition represses tumor growth and remodels immune cell composition. Importantly, inhibition of the cyclin-dependent kinases 4/6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Moreover, in p53-deficient CRC cells, HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR and reduces cancer growth. Likewise, p53-mutated PDOs respond to dual HSF1-HSP90 inhibition, providing a strategy to target CRC independent of the p53 status. In sum, we provide new options to improve HSP90-based therapies to enhance CRC therapies.

PURPOSE:Mutant p53 stabilized by heat shock protein 90 (HSP90) is a novel target in oncology. The open-label, randomized phase II GANNET53 trial is the first to evaluate the HSP90 inhibitor ganetespib (G) with paclitaxel (P) in platinum-resistant epithelial ovarian cancer (EUDRACT 2013-003868-31; EU FP7 #602602). PATIENTS AND METHODS:Patients were randomized 2:1 to receive G + P or P alone until progression. Primary endpoints were progression-free survival (PFS) and PFS rate at 6 months. Exploratory endpoints were biomarkers based on p53 and HSP90. RESULTS:A total of 133 patients were enrolled. The median PFS was 3.5 (G + P) and 5.3 months (P) (HR = 1.3; 95% confidence interval, 0.897-1.895; P = 0.16), and PFS rates at 6 months were 22% (G + P) and 33% (P). No significant differences were found in overall survival, objective response rate, and post-progression PFS between arms. The most frequent adverse events were diarrhea (79% vs. 26%), anemia (46% vs. 51%), nausea (41% vs. 40%), and peripheral neuropathy (36% vs. 47%). Serious adverse events were more common in G + P (39.5% vs. 23.3%). Gastrointestinal perforation was a new safety finding. Despite a high TP53 mutation frequency, HSP90-p53 complexes were detected in only 39.6% of the cases and were also detected stably during treatment. In vitro, no synergistic effects of G + P were observed, and mutant p53 depletion did not sensitize ovarian cancer cells to treatment. CONCLUSIONS:Although no major safety findings were observed, G + P did not lead to survival benefit. Our companion diagnostic program confirmed that G + P do not favorably cooperate in killing ovarian cancer cells.

Disease progression is a major problem in ovarian cancer. There are very few treatment options for patients with platinum-resistant ovarian cancer (PROC), and therefore, these patients have a particularly poor prognosis. The aim of the present study was to identify markers for monitoring the response of 123 PROC patients enrolled in the Phase I/II GANNET53 clinical trial, which evaluated the efficacy of Ganetespib in combination with standard chemotherapy versus standard chemotherapy alone. In total, 474 blood samples were collected, comprising baseline samples taken before the first administration of the study drugs and serial samples taken during treatment until further disease progression (PD). After microfluidic enrichment, 27 gene transcripts were analyzed using quantitative polymerase chain reaction and their utility for disease monitoring was evaluated. At baseline, ERCC1 was associated with an increased risk of PD (hazard ratio [HR] 1.75, 95% confidence interval [CI]: 1.20-2.55; p = 0.005), while baseline CDH1 and ESR1 may have a risk-reducing effect (CDH1 HR 0.66, 95% CI: 0.46-0.96; p = 0.024; ESR1 HR 0.58, 95% CI: 0.39-0.86; p = 0.002). ERCC1 was observed significantly more often (72.7% vs. 53.9%; p = 0.032) and ESR1 significantly less frequently (59.1% vs. 78.3%; p = 0.018) in blood samples taken at radiologically confirmed PD than at controlled disease. At any time during treatment, ERCC1-presence and ESR1-absence were associated with short PFS and with higher odds of PD within 6 months (odds ratio 12.77, 95% CI: 4.08-39.97; p < 0.001). Our study demonstrates the clinical relevance of ESR1 and ERCC1 and may encourage the analysis of liquid biopsy samples for the management of PROC patients.

Clonal hematopoiesis (CH) driven by mutations in the DNA damage response (DDR) pathway is frequent in patients with cancer and is associated with a higher risk of therapy-related myeloid neoplasms (t-MNs). Here, we analyzed 423 serial whole blood and plasma samples from 103 patients with relapsed high-grade ovarian cancer receiving carboplatin, poly(ADP-ribose) polymerase inhibitor (PARPi) and heat shock protein 90 inhibitor (HSP90i) treatment within the phase II EUDARIO trial using error-corrected sequencing of 72 genes. DDR-driven CH was detected in 35% of patients and was associated with longer duration of prior PARPi treatment. TP53- and PPM1D-mutated clones exhibited substantially higher clonal expansion rates than DNMT3A- or TET2-mutated clones during treatment. Expansion of DDR clones correlated with HSP90i exposure across the three study arms and was partially abrogated by the presence of germline mutations related to homologous recombination deficiency. Single-cell DNA sequencing of selected samples revealed clonal exclusivity of DDR mutations, and identified DDR-mutated clones as the origin of t-MN in two investigated cases. Together, these results provide unique insights into the architecture and the preferential selection of DDR-mutated hematopoietic clones under intense DNA-damaging treatment. Specifically, PARPi and HSP90i therapies pose an independent risk for the expansion of DDR-CH in a dose-dependent manner.

Supplementary Figures S1-S3 from Functional Inactivation of Endogenous MDM2 and CHIP by HSP90 Causes Aberrant Stabilization of Mutant p53 in Human Cancer Cells

Supplementary Figure Legends from Mitochondrially Targeted p53 Has Tumor Suppressor Activities <i>In vivo</i>

Clonal hematopoiesis (CH), characterized by the expansion of somatically mutated hematopoietic stem cells, is an age-related phenomenon associated with the development of hematologic malignancies, increased cardiovascular risk, and other age-related proinflammatory conditions. In patients with solid tumors, cytotoxic therapies differentially select for clones with mutations in the DNA damage response (DDR) pathway, i.e. TP53 and PPM1D, leading to an elevated risk for therapy-related myeloid malignancies (t-MNs). Poly(ADP-ribose) polymerase inhibitors (PARPi) are frequently used to treat ovarian cancer with particularly high efficacy reported in tumors exhibiting homologous recombination (HR) deficiency caused by mutations in BRCA1/2 or other HR pathway genes. However, recent data indicate that PARPi treatment is also associated with an elevated risk for t-MNs. Here, we performed an in-depth analysis of CH in whole-blood (WB) and plasma of patients with relapsed high-grade ovarian cancer (HGOC) receiving platinum-based chemotherapy and PARPi treatment, elucidating the mutational landscape of CH, gene-specific dynamics, as well as the potential interference of CH with putative tumor-derived mutations in cell-free DNA (cfDNA). Serial specimen of 103 patients with relapsed, platinum-sensitive HGOC participating in the European Trial on Enhanced DNA Repair Inhibition in Ovarian Cancer (EUDARIO) study (ENGOT-ov48/NCT03783949) were analyzed using error-corrected targeted sequencing with a custom panel covering 45 myeloid and 27 HR-related genes. Study treatment consisted of six cycles of carboplatin-based chemotherapy followed by maintenance therapy with Niraparib ± the HSP90 inhibitor Ganetespib. A total of 128 somatic mutations with variant allele frequencies (VAFs) ≥ 1% were identified in 58 patients (56%) with a high fraction of DDR mutations (51 PPM1D mutations in 27 patients, 7 TP53 mutations in 7 patients). Age (median 62 years) and the number of previous therapy lines (median 1, range 1-5 lines) were significantly associated with the presence of CH. Specifically, prior exposure to PARPi treatment was strongly associated with both the presence of CH and the number of CH mutations. CH-positive patients had significantly shorter progression-free survival (median 7.9 months vs 10.6 months, p = 0.021 in log-rank test), and a trend for shorter overall survival (median 21.1 months vs 27.7 months, p = 0.16) in univariate survival analysis. With respect to adverse events, CH-positive patients more frequently had infections during study treatment (49% vs 26%, p = 0.025). All but 5 CH mutations with VAF ≥ 1% were also detectable in cfDNA with significant correlation of VAFs (R = 0.84, p < 0.0001). Mutations occurring solely in cfDNA (putatively tumor-derived) had a high fraction of TP53 and HR-related mutations, while DNMT3A and PPM1D, were rarely mutated (Figure 1). However, 23/87 (26%) of TP53 mutations and 31/98 (32%) of mutations in HR-related genes detected in cfDNA were of hematopoietic origin, underlining the importance of parallel sequencing of WB in liquid biopsies to avoid false positive results. The dynamics of CH clones under carboplatin and PARPi treatment were assessed in paired WB samples at initiation/end of treatment, which were available for 62 patients. 96 clones that initially had a VAF < 1% emerged during the treatment, including 56 PPM1D- and 10 TP53-mutated clones. Gene-wise comparison revealed a significantly higher median clonal fitness in PPM1D- and TP53-mutated clones compared to TET2- or DNMT3A-mutated clones (Figure 2). PPM1D-mutated clones exhibited lower fitness during PARPi treatment compared to carboplatin treatment (p = 0.0015 in Wilcoxon rank sum test), which was not the case for TP53-mutated clones. Finally, 8 samples with multiple mutations were subjected to single-cell genotyping on the Mission Bio Tapestri platform to investigate their clonal architecture. Preliminary data indicate clonal exclusiveness of co-occurring DDR mutations. In summary, our data reveals a high prevalence of CH in patients with relapsed HGOC and provides novel insights into the clonal architecture and dynamics of CH under carboplatin and PARPi treatment with a differential selection of DDR-driven clones. Moreover, we report a relevant interference of CH-derived mutations with tumor-derived mutations in cfDNA.

Supplementary Figure 7 from Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Supplementary Tables 1 & 2 from Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Supplementary Tables 1-4 from p53-Responsive MicroRNAs 192 and 215 Are Capable of Inducing Cell Cycle Arrest

Supplementary Legends 1-8 from p53-Responsive MicroRNAs 192 and 215 Are Capable of Inducing Cell Cycle Arrest

The NFIA-ETO2 fusion is the product of a t(1;16)(p31;q24) chromosomal translocation, so far, exclusively found in pediatric patients with pure erythroid leukemia (PEL). To address the role for the pathogenesis of the disease, we facilitated the expression of the NFIA-ETO2 fusion in murine erythroblasts (EBs). We observed that NFIA-ETO2 significantly increased proliferation and impaired erythroid differentiation of murine erythroleukemia cells and of primary fetal liver-derived EBs. However, NFIA-ETO2-expressing EBs acquired neither aberrant in vitro clonogenic activity nor disease-inducing potential upon transplantation into irradiated syngenic mice. In contrast, in the presence of 1 of the most prevalent erythroleukemia-associated mutations, TP53R248Q, expression of NFIA-ETO2 resulted in aberrant clonogenic activity and induced a fully penetrant transplantable PEL-like disease in mice. Molecular studies support that NFIA-ETO2 interferes with erythroid differentiation by preferentially binding and repressing erythroid genes that contain NFI binding sites and/or are decorated by ETO2, resulting in a activity shift from GATA- to ETS-motif-containing target genes. In contrast, TP53R248Q does not affect erythroid differentiation but provides self-renewal and survival potential, mostly via downregulation of known TP53 targets. Collectively, our work indicates that NFIA-ETO2 initiates PEL by suppressing gene expression programs of terminal erythroid differentiation and cooperates with TP53 mutation to induce erythroleukemia.

Supplementary Figure 6 from Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Supplementary Figure 8 from Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Supplementary Table 4C from Mitochondrially Targeted p53 Has Tumor Suppressor Activities <i>In vivo</i>

Supplementary Table 4B from Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

TP53-mutant acute myeloid leukemia (AML) remains the ultimate therapeutic challenge. Epichaperomes, formed in malignant cells, consist of heat shock protein 90 (HSP90) and associated proteins that support the maturation, activity, and stability of oncogenic kinases and transcription factors including mutant p53. High-throughput drug screening identified HSP90 inhibitors as top hits in isogenic TP53-wild-type (WT) and-mutant AML cells. We detected epichaperomes in AML cells and stem/progenitor cells with TP53 mutations but not in healthy bone marrow (BM) cells. Hence, we investigated the ther-apeutic potential of specifically targeting epichaperomes with PU-H71 in TP53-mutant AML based on its preferred binding to HSP90 within epichaperomes. PU-H71 effectively suppressed cell intrinsic stress responses and killed AML cells, primarily by inducing apoptosis; targeted TP53-mutant stem/progenitor cells; and prolonged survival of TP53-mutant AML xenograft and patient-derived xenograft models, but it had minimal effects on healthy human BM CD34(+) cells or on murine hematopoiesis. PU-H71 decreased MCL-1 and multiple signal proteins, increased proapoptotic Bcl-2-like protein 11 levels, and synergized with BCL-2 inhibitor venetoclax in TP53-mutant AML. Notably, PU-H71 effectively killed TP53-WT and-mutant cells in isogenic TP53-WT/TP53-R248W Molm13 cell mixtures, whereas MDM2 or BCL-2 inhibition only reduced TP53-WT but favored the outgrowth of TP53-mutant cells. Venetoclax enhanced the killing of both TP53-WT and-mutant cells by PU-H71 in a xenograft model. Our data suggest that epichaperome function is essential for TP53-mutant AML growth and survival and that its inhibition targets mutant AML and stem/progenitor cells, enhances venetoclax activity, and prevents the outgrowth of venetoclax-resistant TP53-mutant AML clones. These concepts warrant clinical evaluation.