Interferon-ε is a tumour suppressor and restricts ovarian cancer.

High-grade serous ovarian cancers have low survival rates because of their late presentation with extensive peritoneal metastases and frequent chemoresistance1, and require new treatments guided by novel insights into pathogenesis. Here we describe the intrinsic tumour-suppressive activities of interferon-ε (IFNε). IFNε is constitutively expressed in epithelial cells of the fallopian tube, the cell of origin of high-grade serous ovarian cancers, and is then lost during development of these tumours. We characterize its anti-tumour activity in several preclinical models: ovarian cancer patient-derived xenografts, orthotopic and disseminated syngeneic models, and tumour cell lines with or without mutations in Trp53 and Brca genes. We use manipulation of the IFNε receptor IFNAR1 in different cell compartments, differential exposure status to IFNε and global measures of IFN signalling to show that the mechanism of the anti-tumour activity of IFNε involves direct action on tumour cells and, crucially, activation of anti-tumour immunity. IFNε activated anti-tumour T and natural killer cells and prevented the accumulation and activation of myeloid-derived suppressor cells and regulatory T cells. Thus, we demonstrate that IFNε is an intrinsic tumour suppressor in the female reproductive tract whose activities in models of established and advanced ovarian cancer, distinct from other type I IFNs, are compelling indications of potential new therapeutic approaches for ovarian cancer.

A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors.

BACKGROUND: We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors. METHODS: The levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models. RESULTS: Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively. CONCLUSION: A clinical trial may be justified to further investigate the utility of IAP inhibitors.

Development and survival of MYC-driven lymphomas require the MYC antagonist MNT to curb MYC-induced apoptosis.

Deregulated overexpression of MYC is implicated in the development and malignant progression of most (∼70%) human tumors. MYC drives cell growth and proliferation, but also, at high levels, promotes apoptosis. Here, we report that the proliferative capacity of MYC-driven normal and neoplastic B lymphoid cells depends on MNT, a MYC-related transcriptional repressor. Our genetic data establish that MNT synergizes with MYC by suppressing MYC-driven apoptosis, and that it does so primarily by reducing the level of pro-apoptotic BIM. In Eµ-Myc mice, which model the MYC/IGH chromosome translocation in Burkitt's lymphoma, homozygous Mnt deletion greatly reduced lymphoma incidence by enhancing apoptosis and markedly decreasing premalignant B lymphoid cell populations. Strikingly, by inducing Mnt deletion within transplanted fully malignant Eµ-Myc lymphoma cells, we significantly extended transplant recipient survival. The dependency of lymphomas on MNT for survival suggests that drugs inhibiting MNT could significantly boost therapy of MYC-driven tumors by enhancing intrinsic MYC-driven apoptosis.

DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa.

Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocyte nuclei and is essential for their apoptosis triggered by DNA damage in vivo. After γ-irradiation, induction of the proapoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from WT and Trp53(-/-) mice but not in those from TAp63-deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women's health.

Apoptosis-promoted tumorigenesis: gamma-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death.

Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in gamma-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from gamma-irradiation-induced death, because their glucocorticoid-mediated decimation in Puma-deficient mice activated cycling of stem/progenitor cells and restored thymic lymphomagenesis. Our demonstration that cycles of cell attrition and repopulation by stem/progenitor cells can drive tumorigenesis has parallels in human cancers, such as therapy-induced malignancies.

Plasmacytomagenesis in Eµ-v-abl transgenic mice is accelerated when apoptosis is restrained.

Mice susceptible to plasma cell tumors provide a useful model for human multiple myeloma. We previously showed that mice expressing an Eµ-v-abl oncogene solely develop plasmacytomas. Here we show that loss of the proapoptotic BH3-only protein Bim or, to a lesser extent, overexpression of antiapoptotic Bcl-2 or Mcl-1, significantly accelerated the development of plasmacytomas and increased their incidence. Disease was preceded by an increased abundance of plasma cells, presumably reflecting their enhanced survival capacity in vivo. Plasmacytomas of each genotype expressed high levels of v-abl and frequently harbored a rearranged c-myc gene, probably as a result of chromosome translocation. As in human multiple myelomas, elevated expression of cyclin D genes was common, and p53 deregulation was rare. Our results for plasmacytomas highlight the significance of antiapoptotic changes in multiple myeloma, which include elevated expression of Mcl-1 and, less frequently, Bcl-2, and suggest that closer attention to defects in Bim expression is warranted.

ABT-199, a new Bcl-2-specific BH3 mimetic, has in vivo efficacy against aggressive Myc-driven mouse lymphomas without provoking thrombocytopenia.

BH3-only proteins trigger the stress apoptosis pathway and chemical mimetics have great potential for cancer therapy. BH3-only proteins inhibit antiapoptotic members of the Bcl-2 family. Promising BH3 mimetic ABT-737 and the related orally available compound ABT-263 (navitoclax) bind avidly to antiapoptotic Bcl-2, Bcl-xL, and Bcl-w. However, their interaction with Bcl-xL provokes thrombocytopenia, which has proven to be the dose-limiting toxicity. We have tested the efficacy of ABT-199, a new Bcl-2-specific BH3 mimetic, against aggressive progenitor cell lymphomas derived from bitransgenic myc/bcl-2 mice. As a single agent, ABT-199 was as effective as ABT-737 in prolonging survival of immunocompetent tumor-bearing mice without causing thrombocytopenia. Both drugs acted rapidly but, contrary to prevailing models, their apoptotic activity did not rely upon the BH3-only protein Bim. When ABT-737 was combined with the proteosome inhibitor bortezomib or CDK inhibitor purvalanol, many treated animals achieved long-term remission.

Bcl-2, Bcl-x(L), and Bcl-w are not equivalent targets of ABT-737 and navitoclax (ABT-263) in lymphoid and leukemic cells.

The BH3-mimetic ABT-737 and an orally bioavailable compound of the same class, navitoclax (ABT-263), have shown promising antitumor efficacy in preclinical and early clinical studies. Although both drugs avidly bind Bcl-2, Bcl-x(L), and Bcl-w in vitro, we find that Bcl-2 is the critical target in vivo, suggesting that patients with tumors overexpressing Bcl-2 will probably benefit. In human non-Hodgkin lymphomas, high expression of Bcl-2 but not Bcl-x(L) predicted sensitivity to ABT-263. Moreover, we show that increasing Bcl-2 sensitized normal and transformed lymphoid cells to ABT-737 by elevating proapoptotic Bim. In striking contrast, increasing Bcl-x(L) or Bcl-w conferred robust resistance to ABT-737, despite also increasing Bim. Cell-based protein redistribution assays unexpectedly revealed that ABT-737 disrupts Bcl-2/Bim complexes more readily than Bcl-x(L)/Bim or Bcl-w/Bim complexes. These results have profound implications for how BH3-mimetics induce apoptosis and how the use of these compounds can be optimized for treating lymphoid malignancies.

The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized.

Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects ABT-737's inability to target another prosurvival relative, Mcl-1. Downregulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in a mouse lymphoma model conferred resistance. In contrast, cells overexpressing Bcl-2 remained highly sensitive to ABT-737. Hence, ABT-737 should prove efficacious in tumors with low Mcl-1 levels, or when combined with agents that inactivate Mcl-1, even to treat those tumors that overexpress Bcl-2.

The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair.

BRIP1 (also called BACH1) is a DEAH helicase that interacts with the BRCT domain of BRCA1 (refs. 1-6) and has an important role in BRCA1-dependent DNA repair and checkpoint functions. We cloned the chicken ortholog of BRIP1 and established a homozygous knockout in the avian B-cell line DT40. The phenotype of these brip1 mutant cells in response to DNA damage differs from that of brca1 mutant cells and more closely resembles that of fancc mutant cells, with a profound sensitivity to the DNA-crosslinking agent cisplatin and acute cell-cycle arrest in late S-G2 phase. These defects are corrected by expression of human BRIP1 lacking the BRCT-interaction domain. Moreover, in human cells exposed to mitomycin C, short interfering RNA-mediated knock-down of BRIP1 leads to a substantial increase in chromosome aberrations, a characteristic phenotype of cells derived from individuals with Fanconi anemia. Because brip1 mutant cells are proficient for ubiquitination of FANCD2 protein, our data indicate that BRIP1 has a function in the Fanconi anemia pathway that is independent of BRCA1 and downstream of FANCD2 activation.

CHK1 inhibitor SRA737 is active in PARP inhibitor resistant and CCNE1 amplified ovarian cancer.

High-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) are initially responsive to poly (ADP-ribose) polymerase inhibitors (PARPi), but resistance ultimately emerges. HGSOC with CCNE1 amplification (CCNE1 amp) are associated with resistance to PARPi and platinum treatments. High replication stress in HRD and CCNE1 amp HGSOC leads to increased reliance on checkpoint kinase 1 (CHK1), a key regulator of cell cycle progression and the replication stress response. Here, we investigated the anti-tumor activity of the potent, highly selective, orally bioavailable CHK1 inhibitor (CHK1i), SRA737, in both acquired PARPi-resistant BRCA1/2 mutant and CCNE1 amp HGSOC models. We demonstrated that SRA737 increased replication stress and induced subsequent cell death in vitro. SRA737 monotherapy in vivo prolonged survival in CCNE1 amp models, suggesting a potential biomarker for CHK1i therapy. Combination SRA737 and PARPi therapy increased tumor regression in both PARPi-resistant and CCNE1 amp patient-derived xenograft models, warranting further study in these HGSOC subgroups.

MNT suppresses T cell apoptosis via BIM and is critical for T lymphomagenesis.

The importance of c-MYC in regulating lymphopoiesis and promoting lymphomagenesis is well-established. Far less appreciated is the vital supporting role of MYC's relative MNT. Using Rag1Cre-mediated Mnt deletion in lymphoid progenitor cells, we show here that, during normal T cell development, MNT loss enhances apoptosis, at least in part by elevating expression of the pro-apoptotic BH3-only protein BIM. Moreover, using T lymphoma-prone VavP-MYC transgenic mice, we show that Mnt deletion reduces the pool of pre-malignant MYC-driven T lymphoid cells and abrogates thymic T lymphomagenesis. In addition, we establish that Mnt deletion prevents T lymphoma development in γ-irradiated mice, most likely by enhancing apoptosis of T lymphoid cells repopulating the depleted thymus. Taken together with our recent demonstration that MNT is vital for the survival of MYC-driven pre-malignant and malignant B lymphoid cells, these results suggest that MNT represents an important new drug target for both T and B lymphoid malignancies.

The microtubule inhibitor eribulin demonstrates efficacy in platinum-resistant and refractory high-grade serous ovarian cancer patient-derived xenograft models.

Background: Despite initial response to platinum-based chemotherapy and PARP inhibitor therapy (PARPi), nearly all recurrent high-grade serous ovarian cancer (HGSC) will acquire lethal drug resistance; indeed, ~15% of individuals have de novo platinum-refractory disease. Objectives: To determine the potential of anti-microtubule agent (AMA) therapy (paclitaxel, vinorelbine and eribulin) in platinum-resistant or refractory (PRR) HGSC by assessing response in patient-derived xenograft (PDX) models of HGSC. Design and methods: Of 13 PRR HGSC PDX, six were primary PRR, derived from chemotherapy-naïve samples (one was BRCA2 mutant) and seven were from samples obtained following chemotherapy treatment in the clinic (five were mutant for either BRCA1 or BRCA2 (BRCA1/2), four with prior PARPi exposure), recapitulating the population of individuals with aggressive treatment-resistant HGSC in the clinic. Molecular analyses and in vivo treatment studies were undertaken. Results: Seven out of thirteen PRR PDX (54%) were sensitive to treatment with the AMA, eribulin (time to progressive disease (PD) ⩾100 days from the start of treatment) and 11 out of 13 PDX (85%) derived significant benefit from eribulin [time to harvest (TTH) for each PDX with p < 0.002]. In 5 out of 10 platinum-refractory HGSC PDX (50%) and one out of three platinum-resistant PDX (33%), eribulin was more efficacious than was cisplatin, with longer time to PD and significantly extended TTH (each PDX p < 0.02). Furthermore, four of these models were extremely sensitive to all three AMA tested, maintaining response until the end of the experiment (120d post-treatment start). Despite harbouring secondary BRCA2 mutations, two BRCA2-mutant PDX models derived from heavily pre-treated individuals were sensitive to AMA. PRR HGSC PDX models showing greater sensitivity to AMA had high proliferative indices and oncogene expression. Two PDX models, both with prior chemotherapy and/or PARPi exposure, were refractory to all AMA, one of which harboured the SLC25A40-ABCB1 fusion, known to upregulate drug efflux via MDR1. Conclusion: The efficacy observed for eribulin in PRR HGSC PDX was similar to that observed for paclitaxel, which transformed ovarian cancer clinical practice. Eribulin is therefore worthy of further consideration in clinical trials, particularly in ovarian carcinoma with early failure of carboplatin/paclitaxel chemotherapy.

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.

Targeting homologous recombination deficiency in uterine leiomyosarcoma.

BACKGROUND: Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of < 10%. Mutations in the homologous recombination (HR) DNA repair pathway have been observed in ~ 10% of uLMS cases, with reports of some individuals benefiting from poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) therapy, which targets this DNA repair defect. In this report, we screened individuals with uLMS, accrued nationally, for mutations in the HR repair pathway and explored new approaches to therapeutic targeting. METHODS: A cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting. RESULTS: All 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs. CONCLUSIONS: Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance.

Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eµ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eµ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

Maximal killing of lymphoma cells by DNA damage-inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim.

DNA-damaging chemotherapy is the backbone of cancer treatment, although it is not clear how such treatments kill tumor cells. In nontransformed lymphoid cells, the combined loss of 2 proapoptotic p53 target genes, Puma and Noxa, induces as much resistance to DNA damage as loss of p53 itself. In Eµ-Myc lymphomas, however, lack of both Puma and Noxa resulted in no greater drug resistance than lack of Puma alone. A third B-cell lymphoma-2 homology domain (BH)3-only gene, Bim, although not a direct p53 target, was up-regulated in Eµ-Myc lymphomas incurring DNA damage, and knockdown of Bim levels markedly increased the drug resistance of Eµ-Myc/Puma(-/-)Noxa(-/-) lymphomas both in vitro and in vivo. Remarkably, c-MYC-driven lymphoma cell lines from Noxa(-/-)Puma(-/-)Bim(-/-) mice were as resistant as those lacking p53. Thus, the combinatorial action of Puma, Noxa, and Bim is critical for optimal apoptotic responses of lymphoma cells to 2 commonly used DNA-damaging chemotherapeutic agents, identifying Bim as an additional biomarker for treatment outcome in the clinic.

Myeloid progenitor cells lacking p53 exhibit delayed up-regulation of Puma and prolonged survival after cytokine deprivation.

Loss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)-dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53(-/-), and FoxO3a(-/-) FDM cells and found that p53(-/-) but not FoxO3a(-/-) cells were protected against IL-3 withdrawal. Loss of p21(cip/waf), which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53(-/-) hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53(-/-) cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis.