Biomarkers of treatment benefit with atezolizumab plus vemurafenib plus cobimetinib in BRAFV600 mutation-positive melanoma.

BACKGROUND: The phase III IMspire150 study (NCT02908672) demonstrated significantly improved progression-free survival (PFS) with atezolizumab, vemurafenib, and cobimetinib (atezolizumab group) versus placebo, vemurafenib, and cobimetinib (control group) in patients with BRAFV600-mutated advanced melanoma. We report exploratory biomarker analyses to optimize targeting of patients who are more likely to benefit from triplet combination therapy. PATIENTS AND METHODS: Five hundred fourteen patients were randomized to atezolizumab (n = 256) or control (n = 258). Outcomes were evaluated in subgroups defined by key biomarkers, including programmed death-ligand 1 (PD-L1) expression, lactate dehydrogenase (LDH) level, tumor mutational burden (TMB), and interferon-γ (IFN-γ) gene signature. Exploratory recursive partitioning analysis was then used to model associations between PFS and baseline covariates, including key biomarkers. RESULTS: PFS benefit for atezolizumab versus control was greater in patients with high TMB [≥10 mutations/Mb; hazard ratio (HR) 0.73; 95% confidence interval (CI) 0.52-1.02; P = 0.067] versus low TMB (<10 mutations/Mb; HR 0.92; 95% CI 0.65-1.30; P = 0.64) and similar between patients with strong IFN-γ (≥median; HR 0.76; 95% CI 0.54-1.06) versus weak IFN-γ (

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

Surveillance imaging with FDG-PET/CT in the post-operative follow-up of stage 3 melanoma.

Background: As early detection of recurrent melanoma maximizes treatment options, patients usually undergo post-operative imaging surveillance, increasingly with FDG-PET/CT (PET). To assess this, we evaluated stage 3 melanoma patients who underwent prospectively applied and sub-stage-specific schedules of PET surveillance. Patients and methods: From 2009, patients with stage 3 melanoma routinely underwent PET +/- MRI brain scans via defined schedules based on sub-stage-specific relapse probabilities. Data were collected regarding patient characteristics and outcomes. Contingency analyses were carried out of imaging outcomes. Results: One hundred and seventy patients (stage 3A: 34; 3B: 93; 3C: 43) underwent radiological surveillance. Relapses were identified in 65 (38%) patients, of which 45 (69%) were asymptomatic. False-positive imaging findings occurred in 7%, and 6% had treatable second (non-melanoma) malignancies. Positive predictive values (PPV) of individual scans were 56%-83%. Negative scans had predictive values of 89%-96% for true non-recurrence [negative predictive values (NPV)] until the next scan. A negative PET at 18 months had NPVs of 80%-84% for true non-recurrence at any time in the 47-month (median) follow-up period. Sensitivity and specificity of the overall approach of sub-stage-specific PET surveillance were 70% and 87%, respectively. Of relapsed patients, 33 (52%) underwent potentially curative resection and 10 (16%) remained disease-free after 24 months (median). Conclusions: Application of sub-stage-specific PET in stage 3 melanoma enables asymptomatic detection of most recurrences, has high NPVs that may provide patient reassurance, and is associated with a high rate of detection of resectable and potentially curable disease at relapse.

Vemurafenib in patients with BRAFV600 mutation-positive metastatic melanoma: final overall survival results of the randomized BRIM-3 study.

BACKGROUND: The BRIM-3 trial showed improved progression-free survival (PFS) and overall survival (OS) for vemurafenib compared with dacarbazine in treatment-naive patients with BRAFV600 mutation-positive metastatic melanoma. We present final OS data from BRIM-3. PATIENTS AND METHODS: Patients were randomly assigned in a 1 : 1 ratio to receive vemurafenib (960 mg twice daily) or dacarbazine (1000 mg/m2 every 3 weeks). OS and PFS were co-primary end points. OS was assessed in the intention-to-treat population, with and without censoring of data for dacarbazine patients who crossed over to vemurafenib. RESULTS: Between 4 January 2010 and 16 December 2010, a total of 675 patients were randomized to vemurafenib (n = 337) or dacarbazine (n = 338, of whom 84 crossed over to vemurafenib). At the time of database lock (14 August 2015), median OS, censored at crossover, was significantly longer for vemurafenib than for dacarbazine {13.6 months [95% confidence interval (CI) 12.0-15.4] versus 9.7 months [95% CI 7.9-12.8; hazard ratio (HR) 0.81 [95% CI 0.67-0.98]; P = 0.03}, as was median OS without censoring at crossover [13.6 months (95% CI 12.0-15.4) versus 10.3 months (95% CI 9.1-12.8); HR 0.81 (95% CI 0.68-0.96); P = 0.01]. Kaplan-Meier estimates of OS rates for vemurafenib versus dacarbazine were 56% versus 46%, 30% versus 24%, 21% versus 19% and 17% versus 16% at 1, 2, 3 and 4 years, respectively. Overall, 173 of the 338 patients (51%) in the dacarbazine arm and 175 of the 337 (52%) of those in the vemurafenib arm received subsequent anticancer therapies, most commonly ipilimumab. Safety data were consistent with the primary analysis. CONCLUSIONS: Vemurafenib continues to be associated with improved median OS in the BRIM-3 trial after extended follow-up. OS curves converged after ≈3 years, likely as a result of crossover from dacarbazine to vemurafenib and receipt of subsequent anticancer therapies. CLINICALTRIALS.GOV: NCT01006980.

Incidence, course, and management of toxicities associated with cobimetinib in combination with vemurafenib in the coBRIM study.

Background: In the coBRIM phase III trial, the addition of cobimetinib, an MEK inhibitor, to vemurafenib, a BRAF inhibitor, significantly improved progression-free survival [hazard ratio (HR), 0.58; P < 0.0001] and overall survival (HR, 0.70; P = 0.005) in advanced BRAF-mutated melanoma. Here, we report on the incidence, course, and management of key adverse events (AEs) in the coBRIM study. Patients and methods: Patients were randomly assigned 1:1 to receive vemurafenib (960 mg twice a day) and either cobimetinib (60 mg once a day, 21 days on/7 days off) or placebo. In addition to standard safety evaluations, patients underwent regular ophthalmic, cardiac, and dermatologic surveillance examinations. Results: Of 495 patients recruited to the study, 493 patients received treatment and constituted the safety population (cobimetinib combined with vemurafenib, 247; vemurafenib, 246). At data cut-off (30 September 2015), median follow-up was 18.5 months. Nearly every patient experienced an AE. In patients who received cobimetinib combined with vemurafenib, the frequency of grade ≥3 AEs was higher than in patients who received vemurafenib alone (75% versus 61%). Most AEs, including grade ≥3 AEs, occurred within the first treatment cycle. After the first cycle (28 days), the incidence of common AEs (rash, diarrhoea, photosensitivity, elevated creatine phosphokinase, serous retinopathy, pyrexia, and liver laboratory abnormalities) decreased substantially over time. Most AEs were managed conservatively by supportive care measures, dose modifications of study treatment, and, occasionally, permanent treatment discontinuation. Conclusions: These data indicate that most AEs arising from treatment with cobimetinib combined with vemurafenib generally occur early in the treatment course, are mild or moderate and are manageable by patient monitoring, dose modification and supportive care. ClinicalTrials.gov: NCT01689519.

Vemurafenib in metastatic melanoma patients with brain metastases: an open-label, single-arm, phase 2, multicentre study.

Background: Vemurafenib has shown activity in patients with BRAFV600 mutated melanoma with brain metastases (BM). This phase 2 study evaluated vemurafenib in patients with/without prior treatment for BM. Methods: Patients with BRAFV600 mutated melanoma with BM were enrolled into cohort 1 (previously untreated BM) and cohort 2 (previously treated BM) and received vemurafenib (960 mg BID) until disease progression (PD) or intolerance. Primary endpoint was best overall response rate (BORR) in the brain in cohort 1 that was evaluated using modified RECIST 1.1 criteria using lesions ≥0.5 cm to assess response. Results: 146 patients were treated (cohort 1 n = 90; cohort 2 n = 56), 62% of whom were male. Median (range) time since diagnosis of BM: 1.0 (0-9) month in cohort 1 and 4.2 (1-68) months in cohort 2. Median duration of treatment was 4.1 months (range 0.3-34.5) in cohort 1 and 4.1 months (range 0.2-27.6) in cohort 2. Intracranial BORR in cohort 1 by an independent review committee (IRC) was 18% (2 CRs, 14 PRs). Extracranial BORR by IRC was 33% in cohort 1 and 23% in cohort 2. Median PFS (brain only, investigator-assessed) was 3.7 months (range 0.03-33.4; IQR 1.9-5.6) in cohort 1 and 4.0 months (range 0.3-27.4; IQR 2.2-7.4) in cohort 2. Median OS was 8.9 months (range 0.6-34.5; IQR 4.9-17.0) in cohort 1 and 9.6 months (range 0.7-34.3; IQR 4.5-18.4) in cohort 2. Adverse events (AEs) were similar in type, grade and frequency to other studies of single-agent vemurafenib. Grade 3/4 AEs occurred in 59 (66%) patients in cohort 1 and 36 (64%) in cohort 2. Overall, 84% of patients died during the study (86% in cohort 1 and 80% in cohort 2), mainly due to disease progression. Conclusions: The study demonstrates clinically meaningful response rates of melanoma BM to vemurafenib, which was well tolerated and without significant CNS toxicity.

Randomized phase II study evaluating veliparib (ABT-888) with temozolomide in patients with metastatic melanoma.

BACKGROUND: Veliparib (ABT-888) is a potent, orally bioavailable, small-molecule inhibitor of the DNA repair enzymes poly ADP-ribose polymerase-1 and -2. Veliparib enhances the efficacy of temozolomide (TMZ) and other cytotoxic agents in preclinical tumor models. PATIENTS AND METHODS: In this multicenter, double-blind trial, adults with unresectable stage III or IV metastatic melanoma were randomized 1:1:1 to TMZ plus veliparib 20 or 40 mg, or placebo twice daily. Efficacy end points included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: Patients (N = 346) were randomized between February 2009 and January 2010. Median [95% confidence interval (CI)] PFS was 3.7 (3.0-5.5), 3.6 (1.9-4.1), and 2 (1.9-3.7) months in the 20-mg, 40-mg, and placebo arms, respectively. Median (95% CI) OS was 10.8 (9.0-13.1), 13.6 (11.4-15.9), and 12.9 (9.8-14.3) months, respectively; ORR was 10.3%, 8.7%, and 7.0%. Exploratory analyses showed patients with low ERCC1 expression had longer PFS when TMZ was combined with veliparib. Toxicities were as expected for TMZ. The frequencies of thrombocytopenia, neutropenia, and leukopenia were significantly increased in the veliparib groups. Grade 3 or 4 adverse events, mainly hematologic toxicities, were seen in 55%, 63%, and 41% of patients in the 20-mg, 40-mg, and placebo arms, respectively. CONCLUSIONS: Median PFS with 20 and 40 mg veliparib almost doubled numerically compared with placebo, but the improvements did not reach statistical significance. OS was not increased with veliparib. Toxicities were similar to TMZ monotherapy, but with increased frequency.

The role of BRAF mutations in primary melanoma growth rate and survival.

BACKGROUND: The clinical behaviour and prognosis of primary melanomas harbouring BRAF mutations is not fully understood. OBJECTIVES: To investigate the effect of mutation status on primary melanoma growth rate and melanoma-specific survival (MSS). METHODS: A prospective cohort of 196 patients with stage I-III primary cutaneous melanoma were followed for a median of 92 months, pre-dating the institution of BRAF inhibitor therapy. Clinicopathological variables were correlated with mutation status and hazard ratios (HRs) estimated for MSS. RESULTS: Of 196 tumours, 77 (39.2%) were BRAF V600E, 10 (5.1%) BRAF V600K and 33 (16.8%) were NRAS mutant. BRAF V600E mutant melanomas were associated with favourable clinical characteristics and tended to be slower growing compared with BRAF V600K, NRAS mutant or BRAF/NRAS wild-type tumours (0.12 mm per month, 0.61 mm per month, 0.36 mm per month and 0.23 mm per month, respectively; P = 0.05). There were 39 melanoma deaths, and BRAF mutant melanomas were associated with poorer MSS in stage I-III disease [HR 2.60, 95% confidence interval (CI) 1.20-5.63; P = 0.02] and stage I-II disease (HR 3.39, 95% CI 1.12-10.22; P = 0.03) after adjusting for other prognostic variables. Considered separately, BRAF V600E mutant melanomas were strongly associated with MSS independently of thickness and nodal status (HR 3.89, 95% CI 1.67-9.09; P < 0.01) but BRAF V600K mutant tumours were not (HR 1.19, 95% CI 0.36-3.92; P = 0.77). CONCLUSIONS: The presence of a BRAF mutation does not necessarily 'drive' more rapid tumour growth but is associated with poorer MSS in patients with early-stage disease.

ATRA and the specific RARα agonist, NRX195183, have opposing effects on the clonogenicity of pre-leukemic murine AML1-ETO bone marrow cells.

All-trans retinoic acid (ATRA) is used successfully in the treatment of acute promyelocytic leukemia (APL). ATRA enhances hematopoietic stem cell self-renewal through retinoic acid receptor (RAR)γ activation while promoting differentiation of committed myeloid progenitors through RARα activation. Its lack of success in the treatment of non-APL acute myeloid leukemia (AML) may be related to ATRA's non-selectivity for the RARα and RARγ isotypes, and specific RARα activation may be more beneficial in promoting myeloid differentiation. To investigate this hypothesis, the effects of ATRA and the specific RARα agonist NRX195183 was assessed in AML1-ETO (AE)-expressing murine bone marrow (BM) progenitors. ATRA potentiated the in vitro clonogenicity of these cells while NRX195183 had the opposite effect. Morphological and flow cytometric analysis confirmed a predominantly immature myeloid population in the ATRA-treated AE cells while the NRX195183-treated cells demonstrated an increase in the mature myeloid population. Similarly, NRX195183 treatment promoted myeloid differentiation in an AE9a in vivo murine model. In the ATRA-treated AE cells, gene expression analyses revealed functional networks involving SERPINE1 and bone morphogenetic protein 2; AKT phosphorylation was upregulated. Collectively, these findings confirm the contrasting roles of specific RARα and RARγ activation in the clonogenicity and differentiation of AE cells with potential significant implications in the treatment of non-APL AML using a specific RARα agonist.

Phase III study of nilotinib versus best supportive care with or without a TKI in patients with gastrointestinal stromal tumors resistant to or intolerant of imatinib and sunitinib.

BACKGROUND: This phase III open-label trial investigated the efficacy of nilotinib in patients with advanced gastrointestinal stromal tumors following prior imatinib and sunitinib failure. PATIENTS AND METHODS: Patients were randomized 2:1 to nilotinib 400 mg b.i.d. or best supportive care (BSC; BSC without tyrosine kinase inhibitor, BSC+imatinib, or BSC+sunitinib). Primary efficacy end point was progression-free survival (PFS) based on blinded central radiology review (CRR). Patients progressing on BSC could cross over to nilotinib. RESULTS: Two hundred and forty-eight patients enrolled. Median PFS was similar between arms (nilotinib 109 days, BSC 111 days; P=0.56). Local investigator-based intent-to-treat (ITT) analysis showed a significantly longer median PFS with nilotinib (119 versus 70 days; P=0.0007). A trend in longer median overall survival (OS) was noted with nilotinib (332 versus 280 days; P=0.29). Post hoc subset analyses in patients with progression and only one prior regimen each of imatinib and sunitinib revealed a significant difference in median OS of >4 months in favor of nilotinib (405 versus 280 days; P=0.02). Nilotinib was well tolerated. CONCLUSION: In the ITT analysis, no significant difference in PFS was observed between treatment arms based on CRR. In the post hoc subset analyses, nilotinib provided significantly longer median OS.

Efficacy of CHK inhibitors as single agents in MYC-driven lymphoma cells.

CHK1 and CHK2 function as effectors of cell cycle checkpoint arrest following DNA damage. Small molecule inhibitors of CHK proteins are under clinical evaluation in combination with chemotherapeutic agents known to induce DNA damage. We examined whether CHK inhibitors could be effective as single agents in malignant cells with inherent DNA damage because of deregulated expression of the oncogene c-Myc. Eµ-myc lymphoma cells showed a dramatic increase in the extent of DNA damage and DNA damage response (DDR) signalling within 1 h of treatment with CHK1 inhibitors followed by caspase-dependent apoptosis and cell death. In p53 wild-type/ARF null Eµ-myc lymphoma cells, apoptotic cell death was preceded by accumulation of DNA damage and the amount of DNA damage correlated with the extent of cell death. This effect was not observed in normal B cells indicating that DNA damage accumulation following CHK inhibition was specific to Eµ-myc lymphoma cells that exhibit inherent DNA damage because of MYC-induced replication stress. Similar results were obtained with another structurally distinct CHK-inhibitor. Eµ-myc p53 null lymphoma cells were more sensitive to a dual CHK1/CHK2 inhibitor than to a CHK1-specific inhibitor. In all cases, the level of DNA damage following treatment was the most consistent indicator of drug sensitivity. Our results suggest that CHK inhibitors would be beneficial therapeutic agents in MYC-driven cancers. We propose that inhibitors of CHK can act in a synthetically lethal manner in cancers with replication stress as a result of these cancers being reliant on CHK proteins for an effective DDR and cell survival.

A phase I study to determine the safety, pharmacokinetics and pharmacodynamics of a dual VEGFR and FGFR inhibitor, brivanib, in patients with advanced or metastatic solid tumors.

BACKGROUND: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD. RESULTS: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180-800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly ≤1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ≥600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts. CONCLUSION: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses ≤800 mg orally q.d., the recommended phase II study dose.

Clinical responses observed with imatinib or sorafenib in melanoma patients expressing mutations in KIT.

BACKGROUND: Mutations in KIT are more frequent in specific melanoma subtypes, and response to KIT inhibition is likely to depend on the identified mutation. METHODS: A total of 32 patients with metastatic acral or mucosal melanoma were screened for mutations in KIT exons 11, 13 and 17. RESULTS: KIT mutations were found in 38% of mucosal and in 6% of acral melanomas. Three patients were treated with imatinib and one with sorafenib. All four patients responded to treatment, but three have since progressed within the brain. CONCLUSION: The observed clinical responses support further investigation of KIT inhibitors in metastatic melanoma, selected according to KIT mutation status.

KIT immunohistochemistry and mutation status in gastrointestinal stromal tumours (GISTs) evaluated for treatment with imatinib.

AIMS: With the availability of effective but expensive treatment in the form of imatinib, accurate diagnosis of gastrointestinal stromal tumour (GIST) is extremely important. The aims of this study were: to describe the clinicopathological, immunohistochemical and molecular features of cases referred to a cancer centre with a possible diagnosis of GIST; to identify pitfalls in the performance and interpretation of KIT immunohistochemistry; to define the role of KIT mutation testing in making a diagnosis of GIST. METHODS AND RESULTS: Morphological review, KIT immunohistochemistry and mutation testing were performed on all cases referred with a diagnosis of GIST or where the diagnosis was under serious consideration on the basis of KIT immunopositivity with a view to treating with imatinib. Thirty-seven cases met the inclusion criteria. Of these, 26 were classified as GIST and 11 as non-GIST. Most GISTs showed strong diffuse membranous, cytoplasmic or paranuclear KIT immunopositivity. Some non-GISTs demonstrated patchy cytoplasmic KIT immunopositivity related to the immunohistochemical protocol used in the external laboratory, which led to erroneous diagnoses of GIST in nine (24%) cases. KIT mutations involving exons 11 or 9 were identified in 22 (88%) GISTs tested and none of the non-GISTs. CONCLUSIONS: An accurate diagnosis of GIST can be made on clinicopathological and immunohistochemical criteria without the need for mutational analysis in most cases, provided proper attention is paid to the immunohistochemical protocol used and, most importantly, control material. False-positive diagnoses of GIST potentially leading to inappropriate treatment with imatinib are more common than missed diagnoses.

Retinoic acid receptor antagonism in vivo expands the numbers of precursor cells during granulopoiesis.

The role/s of retinoids in granulopoiesis has been recognised for many years, being powerful differentiation inducers. The physiological role/s of retinoic acid receptor (RAR)-mediated signalling during adult haemopoiesis has by contrast proved more elusive. The recent generation of highly specific pan-RAR antagonists has now made possible an assessment of the specific physiological role/s of RAR signalling, allowing the separation for the first time of the RAR and RXR pathways. Mice were treated with AGN194310, a synthetic retinoid that antagonises the physiological function of the three RAR isotypes (alpha, beta, gamma) but does not interact with RXRs. Analyses of the granulocytic lineage using Gr-1, c-Kit and CD11b antibodies, demonstrated that granulocyte numbers were strikingly increased across haemopoietic compartments in all AGN194310-treated mice. A significant increase in the frequency of progenitor cells containing granulocytes was observed in the bone marrow of mice following treatment with AGN194310. In contrast we were not able to detect any differences in cell death of either mature granulocytes or granulocytic progenitors from AGN194310-treated mice compared with control animals. These data demonstrate an essential role for RAR signalling in regulating the numbers of granulocytic precursors in vivo.

Cancer and the genetic revolution.

BACKGROUND: The Human Genome Project is nearing completion, providing us with a detailed description of our molecular makeup. As cancer is a disease induced by changes in genes, the Project will be an important tool to enhance our understanding and improve treatment of cancer. OBJECTIVE: The potential outcomes of the new technologies for cancer prediction, profiling and individualising treatments will be examined and examples provided. DISCUSSION: Evolving technologies will allow clinicians to use the information provided by the Genome Project to identify individuals at high risk of developing cancer and to decide on the best treatment. The information will also give guidance as to the best treatment for an existing cancer.

Targeted deletion of the S-phase-specific Myc antagonist Mad3 sensitizes neuronal and lymphoid cells to radiation-induced apoptosis.

The Mad family comprises four basic-helix-loop-helix/leucine zipper proteins, Mad1, Mxi1, Mad3, and Mad4, which heterodimerize with Max and function as transcriptional repressors. The balance between Myc-Max and Mad-Max complexes has been postulated to influence cell proliferation and differentiation. The expression patterns of Mad family genes are complex, but in general, the induction of most family members is linked to cell cycle exit and differentiation. The expression pattern of mad3 is unusual in that mad3 mRNA and protein were found to be restricted to proliferating cells prior to differentiation. We show here that during murine development mad3 is specifically expressed in the S phase of the cell cycle in neuronal progenitor cells that are committed to differentiation. To investigate mad3 function, we disrupted the mad3 gene by homologous recombination in mice. No defect in cell cycle exit and differentiation could be detected in mad3 homozygous mutant mice. However, upon gamma irradiation, increased cell death of thymocytes and neural progenitor cells was observed, implicating mad3 in the regulation of the cellular response to DNA damage.

Analysis of Myc/Max/Mad network members in adipogenesis: inhibition of the proliferative burst and differentiation by ectopically expressed Mad1.

Transcription factors of the Myc/Max/Mad network affect multiple aspects of cellular behavior, including proliferation, differentiation, and apoptosis. Recent studies have shown that Mad proteins can inhibit cellular growth and transformation and thus antagonize the function of Myc proteins. To define further the contribution of these proteins to cellular growth control, we have studied the expression of the respective genes and proteins in 3T3-L1 cells, both upon serum stimulation of quiescent cells and during adipocytic differentiation in response to insulin, dexamethasone, and isobutylmethylxanthine. We found distinct expression patterns for the mad genes. Mad4 was induced when cells exit the cell cycle and, together with mad1, during the late phase of differentiation. In contrast, mad3 expression was associated with progression through S phase and the proliferative burst of differentiating preadipocytes, overlapping in part c-myc expression. DNA binding analyses revealed that the most prominent network complex both in cycling and in differentiating cells was Mnt/Max, whereas c-Myc/Max complexes were detectable only during peak c-Myc expression periods. Ectopic expression of Mad1 in preadipocytes resulted in the inhibition of S phase and the proliferation associated with the proliferative burst; as a consequence, adipocytic differentiation was significantly inhibited. Our findings suggest that the precise temporal regulation of Myc/Max/Mad network proteins is critical for determining cellular behavior.

Dwarfism and dysregulated proliferation in mice overexpressing the MYC antagonist MAD1.

The four members of the MAD family are bHLHZip proteins that heterodimerize with MAX and act as transcriptional repressors. The switch from MYC-MAX complexes to MAD-MAX complexes has been postulated to couple cell-cycle arrest with differentiation. The ectopic expression of Mad1 in transgenic mice led to early postnatal lethality and dwarfism and had a profound inhibitory effect on the proliferation of the hematopoietic cells and embryonic fibroblasts derived from these animals. Compared to wild-type cells, Mad1 transgenic fibroblasts arrested with altered morphology and reduced density at confluence, cycled more slowly, and were delayed in their progression from G0 to the S phase. These changes were accompanied by accumulation of hypophosphorylated retinoblastoma protein and p130. Cyclin D1-associated kinase activity was dramatically reduced in MAD1-overexpressing fibroblasts. However, wild-type cell-cycle distribution and morphology could be rescued in the Mad1 transgenic cells by the introduction of HPV-E7, but not an E7 mutant incapable of binding to pocket proteins. This indicates that the activities of the retinoblastoma family members, via the cyclin D pathway, are likely to be the major targets for MAD1-mediated inhibition of proliferation in primary mouse fibroblasts.