Faculty Bibliography

Background: PP6C binds to regulatory units toaffect a number of important pathways including cell proliferation and DNA repair. Recently two independent groups reported for the first time the presence of somatic mutations in the PPP6C gene in ~10% of short term cultures and limited number of human melanoma tissues. However, the clinical or biological relevance of PPP6C mutations in melanoma patients is unknown. Our objectives were to examine the clinical relevance of PPP6C mutations in a well characterized cohort of melanoma specimens linked to extensive, prospectively-collected clinical information and to explore the functional consequence of different categories of mutations. Methods: Sanger dideoxy sequencing was performed on PCR-amplified DNA from macro-dissected FFPE tumors. Associations between PPP6C mutations and baseline characteristics, recurrence, survival, and BRAF/ NRAS mutational status were examined. The impact of mutations on binding PP6C regulatory units was assessed as well as the effect on additional downstream pathways. Results: 308 primary melanoma patients (118 Stage I, 92 Stage II, and 98 Stage III) were examined (median follow up: 5.3 years). 50 PPP6C mutations in 33 patients (10.7%) were identified with 11 tumors harboring more than one mutation. One mutation (R301C) was identified in 6 patients. PPP6C mutations occurred with similar frequencies across stages and showed no association with BRAF or NRAS mutations.Mutations were categorized into 3 groups: Mutations resulting in premature stop codon (n=9), those occurring in the active site (n=16) and others (n=8). 8/9 (89%) patients with stop mutations recurred and developed visceral metastases. Functional studies revealed that PPP6C mutants also behaved differently; some PPP6C mutations led to decreased binding to regulatory subunits, others, including the R301C mutation did not. Conclusions: Our data suggest that PPP6C mutation is an early event in melanoma progression and independent of BRAF or NRAS mutations. Data also!

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors.

Preclinical and clinical studies demonstrate the feasibility of treating beta-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human beta-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.We generated lentiviral vectors carrying the human beta-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human beta-globin through a novel mechanism that links the rate of transcription of the transgenic beta-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34(+) cells isolated from patients affected by beta-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).Our results suggest two major findings. First, we discovered that for the purpose of expressing the beta-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from beta-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

INTRODUCTION: Perifosine, an orally-bioavailable, novel signal transduction modulator has multiple pathway effects including inhibition of Akt, NFkB and activation of JNK. We conducted a phase I/II study of perifosine + bortezomib (Vel) +/-dexamethasone (Dex) which demonstrated encouraging safety and preliminary clinical activity in relapsed and refractory multiple myeloma (MM) patients (pts) (ASH 2009 # 1869). We report final results, including overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) for the treatment of MM pts previously relapsed from and/or refractory to Vel. METHODS: The phase I stage of the study enrolled a total of 18 pts with a selected phase II dose of perifosine 50 mg qd + Vel 1.3 mg/m² (d 1, 4, 8, 11) in 21-d cycles, which enrolled 66 pts. Dex 20mg (on day of and after each Vel dose) could be added in pts with progressive disease (PD). NCI CTCAE v3.0 was used for toxicity assessment. Response was assessed by modified EBMT and Uniform Criteria. RESULTS: A total of 84 pts were enrolled comprised of 51 men and 33 women, median age 63 y (range 35-89): 88% of pts had relapsed and refractory MM (73% Vel refractory), with a median of 5 lines of prior treatment (range 1-13). Prior therapy included Vel (100%), dex (98%), lenalidomide (76%), thalidomide (75%) and SCT (58%), with pts receiving a median of 2 prior Vel-based treatments. Median interval between diagnosis and inclusion was 4.5 years. Seventy three pts were evaluable for response (having completed at least two or more cycles of therapy), and all 84 were assessed for safety. Most common grade 1/2 events included nausea, diarrhea, fatigue and musculoskeletal pain, which were manageable with supportive care and dose reductions. Grade 3/4 adverse events included thrombocytopenia, neutropenia, anemia, pneumonia and musculoskeletal pain. Two pts had grade 3 peripheral neuropathy (PN), which was reversible with Vel dose reduction and/or treatment discontinuation. No grade 4 PN was observed. Overall 26 (31%) pts had treatment-related PN of any grade. Two pts had perifosine reduced to 50 mg (for GI related toxicity) in the phase 1 portion of the study, and 8 pts reduced to qod dosing in the phase II: 23 pts had Vel dose reductions primarily due to hematologic toxicity. No treatment-related mortality was seen. Eleven pts were inevaluable for response due to unrelated toxicity (n=5), rapid disease progression (< 1 cycle received; n=3), or withdrawal of consent (n=3). For 73 response-evaluable pts, the ORR was 41%, including 4% CR, 18% PR and 19% MR. Prior best response (>= PR) to last Vel-based regimen was 43% (36/84 pts), of which most were triple drug combinations (eg RVD). Of 53 pts who were refractory to prior Vel, ORR to perifosine, Vel +/-Dex was 32%, including 2% CR, 11% PR and 19% MR. ORR for 20 Vel-relapsed pts was 65%; 10% achieved a CR and 35% a PR. Median PFS was 6.4 mos with a range of 5 wks to over 3 yrs (95% CI: 5.3, 7.1), and 8.0 mos (95% CI: 6.4, 9.8) for the 30 evaluable pts who achieved >= MR. Median PFS for the 53 Vel-refractory pts was 5.7 mos (95% CI: 4.3, 6.4), with 40% of pts achieving PFS of > 6 mos. For the 20 Vel-relapsed pts, median PFS was 8.8 mos (95% CI: 6.3, 11.2), with 70% of pts achieving PFS of > 6 mos. Importantly, median PFS for all pts receiving prior Vel-based regimens prior to study entry was 5.3 mos. As of August 2011, median OS for all evaluable study pts was 25 mos (95% CI: 16.3, 31.1), 22.5 mos (95% CI: 14.2, 31.1) for the Vel-refractory pts and 30.4 mos (95% CI: 17.8, NR) for Vel-relapsed pts. Median OS for those pts who achieved PR + CR was 37 mos (95% CI: 13.8, NR) and for those pts who achieved SD + MR was 29 months (95% CI: 18, 32.4). CONCLUSIONS: Perifosine in combination with Vel (+/-dex) demonstrated impressive and durable activity in both heavily pre-treated, Vel-refractory pts (with an ORR of 32% and median OS of 22.5 mos), and in Vel-relapsed pts (with an ORR of 65%, median PFS of 8.8 mos and OS of 30.4 mos), with manageable toxicity. Survival data were encouraging, even in pts exposed to and refractory to Vel, as well as prior IMiDs, so providing another example of a rational combination strategy integrating novel therapies to further improve patient outcome. An international phase III randomized trial evaluating perifosine vs. placebo when combined with Vel and Dex in Vel-exposed pts with relapsed and refractory MM has been granted special protocol assessment (SPA) status and is currently underway to confirm the efficacy of this approach

The Myc transcription factor plays a vital role in both normal cellular physiology and in many human cancers. We have recently demonstrated that nonsense-mediated RNA decay (NMD), a mechanism that rapidly degrades select mRNAs, is inhibited by the stress-induced phosphorylation of translation initiation factor eIF2alpha, and this inhibition stabilizes many transcripts necessary for tumorigenesis. Here, we demonstrate that NMD is inhibited by high Myc expression. We show that the phosphorylation of eIF2alpha, likely due to the ability of Myc to generate reactive oxygen species and augment endoplasmic reticulum stress, is necessary for the inhibition of NMD by Myc. The inhibition of NMD both stabilizes and up-regulates multiple Myc targets, suggesting that the inhibition of NMD may play an important role in the dynamic regulation of genes by Myc

Rationale: Human atherosclerotic plaques contain large numbers of cells deprived of O(2). In murine atherosclerosis, because the plaques are small, it is controversial whether hypoxia can occur. Objective: To examine if murine plaques contain hypoxic cells, and whether hypoxia regulates changes in cellular lipid metabolism and gene expression in macrophages. Methods and Results: Aortic plaques from apolipoprotein-E-deficient mice were immunopositive for hypoxia-inducible transcription factor (HIF-1alpha) and some of its downstream targets. Murine J774 macrophages rendered hypoxic demonstrated significant increases in cellular sterol and triglycerides. The increase in sterol content in hypoxic macrophages correlated with elevated 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity and mRNA levels. In addition, when macrophages were incubated with cholesterol complexes, hypoxic cells accumulated 120% more cholesterol, predominately in the free form. Cholesterol-efflux assays showed that hypoxia significantly decreased efflux mediated by ATP-binding cassette subfamily A member 1 (ABCA1), whose sub cellular localization was altered in both J774 and primary macrophages. Furthermore, in vivo expression patterns of selected genes from cells in hypoxic regions of murine plaques were similar to those from J774 and primary macrophages incubated in hypoxia. The hypoxia-induced accumulation of sterol and decreased cholesterol efflux was substantially reversed in vitro by reducing the expression of the hypoxia-inducible transcription factor, HIF-1alpha. Conclusion: Hypoxic regions are present in murine plaques. Hypoxic macrophages have increased sterol content due to the induction of sterol synthesis and the suppression of cholesterol efflux, effects that are in part mediated by HIF-1alpha

While nonsense-mediated RNA decay (NMD) is an established mechanism to rapidly degrade select transcripts, the physiological regulation and biological significance of NMD are not well characterized. We previously demonstrated that NMD is inhibited in hypoxic cells. Here we show that the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) translation initiation factor by a variety of cellular stresses leads to the inhibition of NMD and that eIF2alpha phosphorylation and NMD inhibition occur in tumors. To explore the significance of this NMD regulation, we used an unbiased approach to identify approximately 750 NMD-targeted mRNAs and found that these mRNAs are overrepresented in stress response and tumor-promoting pathways. Consistent with these findings, the inhibition of NMD promotes cellular resistance to endoplasmic reticulum stress and encourages tumor formation. The transcriptional and translational regulations of gene expression by the microenvironment are established mechanisms by which tumor cells adapt to stress. These data indicate that NMD inhibition by the tumor microenvironment is also an important mechanism to dynamically regulate genes critical for the response to cellular stress and tumorigenesis

Two related ER oxidation 1 (ERO1) proteins, ERO1alpha and ERO1beta, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1alpha and ERO1beta. The peak amplitude of calcium transients in homozygous Ero1alpha mutant adult cardiomyocytes was reduced to 42.0 +/- 2.2% (n=10, P</=0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of beta adrenergic blockade (P</=0.01). In addition, mice lacking ERO1alpha were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31+/-0.02 in the mutant (n=20) vs. 0.23+/-0.03 in the wild type (n=18); P</=0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.-Chin, K. T., Kang, G., Qu, J., Gardner, L. B., Coetzee, W. A., Zito, E., Fishman, G. I., Ron, R. The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load