A phase Ib study evaluating the recommended phase II dose, safety, tolerability, and efficacy of mivavotinib in combination with nivolumab in advanced solid tumors.

Mivavotinib (TAK-659/CB-659), a dual SYK/FLT3 inhibitor, reduced immunosuppressive immune cell populations and suppressed tumor growth in combination with anti-PD-1 therapy in cancer models. This dose-escalation/expansion study investigated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of mivavotinib plus nivolumab in patients with advanced solid tumors. Patients received oral mivavotinib 60-100 mg once-daily plus intravenous nivolumab 3 mg/kg on days 1 and 15 in 28-day cycles until disease progression or unacceptable toxicity. The dose-escalation phase evaluated the recommended phase II dose (RP2D; primary endpoint). The expansion phase evaluated overall response rate (primary end point) at the RP2D in patients with triple-negative breast cancer (TNBC). During dose-escalation (n = 24), two dose-limiting toxicities (grade 4 lipase increased and grade 3 pyrexia) occurred in patients who received mivavotinib 80 mg and 100 mg, respectively. The determined RP2D was once-daily mivavotinib 80 mg plus nivolumab 3 mg/kg. The expansion phase was terminated at ~50% enrollment (n = 17) after failing to meet an ad hoc efficacy futility threshold. Among all 41 patients, common treatment-emergent adverse events (TEAEs) included dyspnea (48.8%), aspartate aminotransferase increased, and pyrexia (46.3% each). Common grade ≥3 TEAEs were hypophosphatemia and anemia (26.8% each). Mivavotinib plasma exposure was generally dose-proportional (60-100 mg). One patient had a partial response. Mivavotinib 80 mg plus nivolumab 3 mg/kg was well tolerated with no new safety signals beyond those of single-agent mivavotinib or nivolumab. Low response rates highlight the challenges of treating unresponsive tumor types, such as TNBC, with this combination and immunotherapies in general. TRIAL REGISTRATION ID: NCT02834247.

CDC7 inhibition induces replication stress-mediated aneuploid cells with an inflammatory phenotype sensitizing tumors to immune checkpoint blockade.

Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.

A phase Ib trial of mivavotinib (TAK-659), a dual SYK/FLT3 inhibitor, in patients with relapsed/refractory acute myeloid leukemia.

Mivavotinib (TAK-659) is an investigational type 1 tyrosine kinase inhibitor with dual activity against spleen tyrosine kinase (SYK) and FMS-like tyrosine kinase 3 (FLT3). We conducted a phase Ib study to investigate the safety, tolerability, and efficacy of mivavotinib in patients with refractory and/or relapsed (R/R) acute myeloid leukemia (AML). Both daily (QD) and twice daily (BID) dosing regimens were evaluated. A total of 43 patients were enrolled, and there were 5 complete responses (4 with incomplete count recovery). In the QD dosing regimen, the maximum tolerated dose (MTD) was not reached up to 160 mg QD per protocol; 140 mg QD was identified as the recommended phase II dose. In the BID dosing regimen, the MTD was 60 mg BID. Thirty patients (70%) experienced a bleeding event on study; the majority were grades 1 or 2, were resolved without mivavotinib modification, and were not considered related to study treatment. Eleven patients (26%) experienced grade ≥3 bleeding events, which were observed most frequently with the 80 mg BID dose. We conducted platelet aggregation studies to investigate the potential role of mivavotinib-mediated SYK inhibition on platelet function. The bleeding events observed may have been the result of several confounding factors, including AML disease status, associated thrombocytopenia, and high doses of mivavotinib. Overall, these findings indicate that the activity of mivavotinib in R/R AML is modest. Furthermore, any future clinical investigation of this agent should be undertaken with caution, particularly in thrombocytopenic patients, due to the potential bleeding risk of SYK inhibition. ClinicalTrials.gov: NCT02323113.

Tumor cell SYK expression modulates the tumor immune microenvironment composition in human cancer via TNF-α dependent signaling.

BACKGROUND: The expression of SYK in cancer cells has been associated with both tumor promoting and tumor suppressive effects. Despite being proposed as anticancer therapeutic target, the possible role of SYK in modulating local adaptive antitumor immune responses remains uncertain. Using detailed analysis of primary human tumors and in vitro models, we reveal the immunomodulatory effect of SYK protein in human solid cancer. METHODS: We spatially mapped SYK kinase in tumor cells, stromal cells and tumor-infiltrating leukocytes (TILs) in 808 primary non-small cell lung carcinomas (NSCLCs) from two cohorts and in 374 breast carcinomas (BCs) from two independent cohorts. We established the associations of localized SYK with clinicopathologic variables and outcomes. The immunomodulatory role of SYK on tumor cells was assessed using in vitro cytokine stimulation, transcriptomic analysis and selective SYK blockade using a small molecule inhibitor. Functional responses were assessed using cocultures of tumor cells with peripheral blood lymphocytes. T cell responses in baseline and post-treatment biopsies from patients with BC treated with a SYK inhibitor in a phase I clinical trial were also studied. RESULTS: Elevated tumor cell or leukocyte SYK expression was associated with high CD4+ and CD8+ TILs and better outcome in both NSCLC and BC. Tumor cell SYK was associated with oncogenic driver mutations in EGFR or KRAS in lung adenocarcinomas and with triple negative phenotype in BC. In cultured tumor cells, SYK was upregulated by TNFα and required for the TNFα-induced proinflammatory responses and T cell activation. SYK blockade after nivolumab in a phase I clinical trial including three patients with advanced triple negative BC reduced TILs and T cell proliferation. Our work establishes the proinflammatory function of tumor cell SYK in lung and breast cancer. SYK signaling in cultured tumor cells is required for T cell activation and SYK blockade limits adaptive antitumor immune responses and tumor rejection in patients with cancer. CONCLUSIONS: Together, our results establish the immunomodulatory role of SYK expression in human solid tumors. This information could be used to develop novel biomarkers and/or therapeutic strategies.

A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.

Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.

Technique for Retraction of the Fourth Compartment Tendons in Approach to the Wrist Joint.

Most procedures that require exposure of the wrist joint requires us to go through the third and fourth compartment. This is followed by dividing the septum between the 3rd and 4th compartments and retracting the EDC tendons ulnarwards. This can be done by various techniques. Using a form of self-retaining retractor is cumbersome and has to be repeatedly repositioned as the tendons keep slipping out. We describe a novel method to maintain this retraction throughout any wrist procedure.

Functional Outcome of Surgically Managed Perilunate Injuries.

BACKGROUND: Perilunate injuries are rare and quite often missed. We present our experience and outcomes, surgically managing these difficult injuries. METHODS: We analysed data from 14 patients who underwent open reduction and internal fixation of perilunate injuries. All patients underwent open reduction and fixation through the dorsal approach. Fractures were fixed with either K-wires or Headless compression screws. At regular intervals radiographs, range of motion, grip strength, modified Mayo score, Quick DASH score and Lyon wrist scores were collected. RESULTS: The average age of our patients was 29.2. Average time to surgical intervention was 11.3 days. The mean follow-up period was 42.3 months. modified Mayo wrist score, QuickDASH score and Lyon wrist scores were 77.86, 1.62 and 80.86, respectively. Wrist flexion/extension arc was 101.43. Wrist radial/ulnar deviation was 50.0. Mean grip strength was 69.93% of the opposite side. Radiological evidence of wrist arthritis and lunate avascular necrosis was seen in 8 (57.14%) and 4 (28.6%) cases, respectively. CONCLUSION: Early open reduction of perilunate injuries gives reliable results, in spite of radiological evidence of wrist arthritis in a majority of the cases.

Covid 19 and orthopaedic surgery in a large trauma centre in India

BackgroundWe are in the midst of a pandemic caused by the novel SARS-Cov-2 virus. A large percentage of the patients are asymptomatic and hospitals around the world are struggling to restart routine services. We report the results of a universal testing protocol of all patients who underwent orthopaedic surgery in the month of July 2020 in a large orthopaedic speciality hospital in Bangalore, India. MethodsA retrospective study of all patients who underwent orthopaedic surgery in the month of July 2020 at a tertiary care orthopaedic speciality hospital in Bangalore, India. All patients underwent nasopharyngeal swab test before surgery. A questionnaire was used to assess the patient before the RT-PCR nasopharyngeal swab test. Data regarding imaging, investigations and follow up was recorded. ResultsIn the month of July 2020, 168 patients underwent routine nasopharyngeal RT-PCR swab test for COVID-19 prior to planned orthopaedic surgical procedure (Both trauma and elective cases). 16 of the RT-PCR tests were positive. However vascular cases and absolute emergencies were done without a RT - PCR test with PPE and all universal precautions. 11 patients underwent emergency surgery without a RT-PCR test. All 16 cases who were positive were asymptomatic. The asymptomatic positive rate was 9.52%. Of the 11 patients who underwent emergency surgery without a RT-PCR test, only one patient had a positive test post-operatively. ConclusionsRoutine nasopharyngeal RT-PCR testing revealed a high rate of asymptomatic cases. If the RT-PCR test is positive, it is best to defer the case till the test returns negative. All precautions must be taken while performing emergency surgeries. Our algorithm in managing patients has proven to be effective and can be replicated with ease to continue operating and taking care of orthopaedic patients during this pandemic.

Spleen Tyrosine Kinase Inhibitor TAK-659 Prevents Splenomegaly and Tumor Development in a Murine Model of Epstein-Barr Virus-Associated Lymphoma.

Epstein-Barr virus (EBV) is associated with several B and epithelial cell cancers. EBV-encoded latent membrane protein 2A (LMP2A) contributes to cellular transformation by mimicking B cell receptor signaling. LMP2A/MYC double transgenic mice develop splenomegaly and B cell lymphoma much faster than MYC transgenic mice do. In this study, we explored the potential therapeutic efficacy of a novel spleen tyrosine kinase (SYK) and FLT3 inhibitor TAK-659 for development of a treatment option for EBV-associated malignancies. In our transgenic model, TAK-659 treatment totally abrogated splenomegaly and tumor development in LMP2A/MYC mice in both pretumor and tumor cell transfer experiments. TAK-659 treatment killed tumor cells, but not host cells within the spleen and tumors. Furthermore, TAK-659 treatment abrogated metastasis of tumor cells into bone marrow. Our data also show that TAK-659 inhibits SYK phosphorylation and induces apoptosis in LMP2A/MYC tumor cells at low nanomolar concentrations. Therefore, TAK-659 may provide an effective therapeutic option for treatment of LMP2A-positive EBV-associated malignancies and should be explored further in clinical trials.IMPORTANCE The novel SYK and FLT3 inhibitor TAK-659 prevents the enlargement of spleen and tumor development in a mouse model of EBV-associated lymphoma by counteracting the activation of cellular kinase SYK through the viral LMP2A gene by inducing cell death in tumor cells but not in nontumor cells. These findings indicate that TAK-659 may be a very effective nontoxic therapeutic molecule especially for EBV-positive hematologic malignancies.

Anatomical study of anterior supramalleolar artery and its potential application to design a bi-foliate fasciocutaneous flap.

PURPOSE: A further understanding of the anterior supramalleolar artery (ASMA) and its potential applications in reconstructive surgery. MATERIALS AND METHODS: A total of 24 fresh lower limbs from fresh cadavers were injected with red latex for dissection. The type of origin, course, diameter of the pedicle, and the distance between the origin of the ASMA from the anterior tibial artery to the extensor retinaculum (O-R) were recorded. Bi-foliate fasciocutaneous flaps were harvested using the branches of the ASMA. RESULTS: We found four types of origin of the ASMA, and we have accordingly classified them into four types. 10 of them were type A, 7 were type B, 6 were type C and 1 was type D. The mean O-R (origin of ASMA to retinaculum) distance was 2.0 ± 0.8 cm. The diameter of the medial branch (D1), the diameter of the lateral branch (D2), and the diameter of artery stem (D3) (only in type A) were 1.0 ± 0.2 mm, 0.8 ± 0.3 mm, 1.1 ± 0.2 mm, respectively. The mean pedicle length of the lateral flap (L1) and medial flap (L2) were 5.1 ± 1.0 cm and 3.7 ± 0.6 cm, respectively. CONCLUSIONS: The ASMA exists constantly with four different types of origin. Its sizable diameter and lengthy pedicle make it suitable for bi-foliate fasciocutaneous flap transfer.

Pedicled vascularised fibular grafting in a flow-through manner for reconstruction of infected non-union of the tibia with preservation of the peroneal artery: a case report.

We report on an 82-year-old man with an infected non-union of the right tibia in which the peroneal artery was the sole artery supplying the lower leg, owing to peripheral arterial disease. He underwent tibial reconstruction using the pedicled vascularised fibular graft in a flow-through manner (without ligation of either the proximal or distal ends of the peroneal artery) and achieved successful bony union.

Reconstruction of shoulder and elbow function using multiple muscle transfers for cervical spondylotic amyotrophy.

STUDY DESIGN: This was a retrospective study of 8 patients with cervical spondylotic amyotrophy who underwent multiple muscle transfers. OBJECTIVE: The purpose of this study was to evaluate results of multiple muscle transfers about the shoulder and elbow in patients with cervical spondylotic amyotrophy. SUMMARY OF BACKGROUND DATA: Cervical spondylotic amyotrophy is characterized by severe muscle atrophy of the shoulder girdle and elbow. Even after cervical spine surgery, many patients have poor shoulder and elbow function. METHODS: Multiple muscle transfer procedures including the transfer of trapezius, pectoralis major, latissimus dorsi muscles, and the Steindler procedure for reconstruction of shoulder and elbow function were performed in 8 patients with cervical spondylotic amyotrophy. Patients were evaluated at a mean of 18.2 months (range, 5-75 mo). RESULTS: All 8 patients obtained satisfactory functional recovery with improvement of active range of motion without any systemic and local complications within 3 to 6 months postoperatively. Patients at the last follow-up had obtained a mean of 91° of shoulder abduction, 111° of shoulder flexion, 23° of external rotation and 110° of elbow flexion. Disability scores (Japanese version) of the arm, shoulder, and hand improved by a mean of 28 points. CONCLUSION: Multiple muscle transfers can improve shoulder and elbow function in cervical spondylotic amyotrophy, in cases of not only poor outcome after cervical surgery, but also in advanced paralysis. It is a useful set of procedures even in old patients, and provides definitive functional improvement of shoulder and elbow function from 3 to 6 months. LEVEL OF EVIDENCE: 4.

Translational exposure-efficacy modeling to optimize the dose and schedule of taxanes combined with the investigational Aurora A kinase inhibitor MLN8237 (alisertib).

Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure-efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m(2) of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m(2) of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents.

Intraosseous xanthoma of the distal radius - a case report.

An intraosseous xanthoma is a very rare condition. It has an aggressive appearance on radiographs mimicking primary or metastatic malignant bone tumors. We report a case of intraosseous xanthoma of the distal radius in a 51-year-old male with no history of hyperlipidaemia. To the best of our knowledge, this condition has not been reported so far in the wrist and forearm region. The lesion was successfully excised and at last follow-up, there were no signs of recurrence and patient has been symptom-free.

Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress.

Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.

De novo discovery of a gamma-secretase inhibitor response signature using a novel in vivo breast tumor model.

Notch pathway signaling plays a fundamental role in normal biological processes and is frequently deregulated in many cancers. Although several hypotheses regarding cancer subpopulations most likely to respond to therapies targeting the Notch pathway have been proposed, clinical utility of these predictive markers has not been shown. To understand the molecular basis of gamma-secretase inhibitor (GSI) sensitivity in breast cancer, we undertook an unbiased, de novo responder identification study using a novel genetically engineered in vivo breast cancer model. We show that tumors arising from this model are heterogeneous on the levels of gene expression, histopathology, growth rate, expression of Notch pathway markers, and response to GSI treatment. In addition, GSI treatment of this model was associated with inhibition of Hes1 and proliferation markers, indicating that GSI treatment inhibits Notch signaling. We then identified a pretreatment gene expression signature comprising 768 genes that is significantly associated with in vivo GSI efficacy across 99 tumor lines. Pathway analysis showed that the GSI responder signature is enriched for Notch pathway components and inflammation/immune-related genes. These data show the power of this novel in vivo model system for the discovery of biomarkers predictive of response to targeted therapies, and provide a basis for the identification of human breast cancers most likely to be sensitive to GSI treatment.

Array comparative genome hybridization for tumor classification and gene discovery in mouse models of malignant melanoma.

Chromosomal numerical aberrations (CNAs), particularly regional amplifications and deletions, are a hallmark of solid tumor genomes. These genomic alterations carry the potential to convey etiologic and clinical significance by virtue of their clonality within a tumor cell population, their distinctive patterns in relation to tumor staging, and their recurrence across different tumor types. In this study, we showed that array-based comparative genomic hybridization (CGH) analysis of genome-wide CNAs can classify tumors on the basis of differing etiologies and provide mechanistic insights to specific biological processes. In a RAS-induced p19(Arf-/-) mouse model that experienced accelerated melanoma formation after UV exposure, array-CGH analysis was effective in distinguishing phenotypically identical melanomas that differed solely by previous UV exposure. Moreover, classification by array-CGH identified key CNAs unique to each class, including amplification of cyclin-dependent kinase 6 in UV-treated cohort, a finding consistent with our recent report that UVB targets components of the p16(INK4a)-cyclin-dependent kinase-RB pathway in melanoma genesis (K. Kannan, et al., Proc. Natl. Acad. Sci. USA, 21: 2003). These results are the first to establish the utility of array-CGH as a means of etiology-based tumor classification in genetically defined cancer-prone models.

Both products of the mouse Ink4a/Arf locus suppress melanoma formation in vivo.

Deletion of the INK4a/ARF locus at 9p21 is detected with high frequency in human melanoma. Within a short genomic distance, this locus encodes several proteins with established tumor-suppressor roles in a broad spectrum of cancer types. Several lines of evidence support the view that p16INK4a and p19ARF exert the tumor-suppressor activities of this locus, although their relative importance in specific cancer types such as melanoma has been less rigorously documented on the genetic level. Here, we exploit a well-defined mouse model of RAS-induced melanomas to examine the impact of germline p16INK4a or p19ARF nullizygosity on melanoma formation. We demonstrate that loss of either Ink4a/Arf product can cooperate with RAS activation to produce clinically indistinguishable melanomas. In line with the common phenotypic end point, we further show that RAS+ p16INK4a-/- melanomas sustain somatic inactivation of p19ARF-p53 and, correspondingly, that RAS+ p19ARF-/- melanomas experience high-frequency loss of p16INK4a. These genetic studies provide definitive proof that p16INK4a and p19ARF cooperate to suppress the development of melanoma in vivo.

Components of the Rb pathway are critical targets of UV mutagenesis in a murine melanoma model.

Epidemiological studies support a link between melanoma risk and UV exposure early in life, yet the molecular targets of UV's mutagenic actions are not known. By using well characterized murine models of melanoma, we provide genetic and molecular evidence that identifies components of the Rb pathway as the principal targets of UV mutagenesis in murine melanoma development. In a melanoma model driven by H-RAS activation and loss of p19(ARF) function, UV exposure resulted in a marked acceleration in melanoma genesis, with nearly half of these tumors harboring amplification of cyclin-dependent kinase (cdk) 6, whereas none of the melanomas arising in the absence of UV treatment possessed cdk6 amplification. Moreover, UV-induced melanomas showed a strict reciprocal relationship between cdk6 amplification and p16(INK4a) loss, which is consistent with the actions of UV along the Rb pathway. Most significantly, UV exposure had no impact on the kinetics of melanoma driven by H-RAS activation and p16(INK4a) deficiency. Together, these molecular and genetic data identify components of the Rb pathway as critical biological targets of UV-induced mutagenesis in the development of murine melanoma in vivo.

A positive feedback mechanism in the transcriptional activation of Apaf-1 by p53 and the coactivator Zac-1.

p53 exerts its tumor suppressor effects by activating genes involved in cell growth arrest and programmed cell death. The p53 target genes inducing growth arrest are well defined whereas those inducing apoptosis are not fully characterized. Proapoptotic activity of p53 was shown to involve several genes like Bax, Noxa and Puma, which may function in the release of cytochrome c from the mitochondria. Cytochrome c associates with Apaf-1 and caspase 9 to form the apoptosome. Genetic and cellular data indicate that Apaf-1 deficiency abrogates the apoptotic effect of p53 and substitutes for p53 loss in promoting tumor formation. Here we show that Apaf-1, the mammalian homologue of C. elegans CED4, is a direct target of p53 as demonstrated by gel shift analysis of the target site sequence in the presence of p53 and by Apaf-1 promoter-luciferase assays. We also show that the p53 activation of the Apaf-1 luciferase construct can be enhanced by the putative tumor suppressor gene product, Zac-1, a transcription factor that has previously been shown to inhibit cell proliferation. Furthermore, we demonstrate that Zac-1 is a possible direct target of p53 since the sequence upstream to the first coding exon of Zac-1 contains a p53 recognition site and the luciferase construct containing this region is activated by p53. These results suggests the existence of a tightly controlled self amplifying mechanism of transcriptional activation leading to apoptosis by p53.