Luke Whitesell

The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3

IPEX is a fatal disorder characterized by immune dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance (MIM 304930). We present genetic evidence that different mutations of the human gene FOXP3, the ortholog of the gene mutated in scurfy mice (Foxp3), causes IPEX syndrome. Recent linkage analysis studies mapped the gene mutated in IPEX to an interval of 17–20-cM at Xp11.23–Xq13.3 (refs. 1,2).

Heat Shock Factor 1 Is a Powerful Multifaceted Modifier of Carcinogenesis

Heat shock factor 1 (HSF1) is the master regulator of the heat shock response in eukaryotes, a very highly conserved protective mechanism. HSF1 function increases survival under a great many pathophysiological conditions. How it might be involved in malignancy remains largely unexplored. We report that eliminating HSF1 protects mice from tumors induced by mutations of the RAS oncogene or a hot spot mutation in the tumor suppressor p53. In cell culture, HSF1 supports malignant transformation by orchestrating a network of core cellular functions including proliferation, survival, protein synthesis, and glucose metabolism. The striking effects of HSF1 on oncogenic transformation are not limited to mouse systems or tumor initiation; human cancer lines of diverse origins show much greater dependence on HSF1 function to maintain proliferation and survival than their nontransformed counterparts. While it enhances organismal survival and longevity under most circumstances, HSF1 has the opposite effect in supporting the lethal phenomenon of cancer.

P185ERBB2 BINDS TO GRP94 IN VIVO : DISSOCIATION OF THE P185ERBB2/GRP94 HETEROCOMPLEX BY BENZOQUINONE ANSAMYCINS PRECEDES DEPLETION OF P185ERBB2

Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the 100-kDa ansamycin-binding protein is GRP94. Membrane-associated p185 exists in a stable complex with GRP94. GA binding to GRP94 disrupts this complex, leading to degradation of pre-existing p185 protein, and resulting in an altered subcellular distribution of newly synthesized p185.

Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease

Invasive fungal infections are a leading cause of mortality among immunocompromised individuals. Treatment is notoriously difficult with the limited armamentarium of antifungal drugs, whose efficacy is compromised by host toxicity, a limited activity spectrum, or the emergence of drug resistance. We previously established that the molecular chaperone Hsp90 enables the emergence and maintenance of fungal drug resistance. For the most prevalent fungal pathogen of humans, Candida albicans, Hsp90 mediates resistance to azoles, which inhibit ergosterol biosynthesis and are the most widely deployed antifungals in the clinic. For the emerging opportunistic pathogen Aspergillus terreus, Hsp90 is required for basal resistance to echinocandins, which inhibit β(1, 3)-glucan synthesis and are the only new class of antifungals to reach the clinic in decades. Here, we explore the therapeutic potential of Hsp90 inhibitors in fungal disease using a tractable host-model system, larvae of the greater wax moth Galleria mellonella, and a murine model of disseminated disease. Combination therapy with Hsp90 inhibitors that are well tolerated in humans and an azole rescued larvae from lethal C. albicans infections. Combination therapy with an Hsp90 inhibitor and an echinocandin rescued larvae from infections with the most lethal mold, Aspergillus fumigatus. In a murine model of disseminated candidiasis, genetic compromise of C. albicans HSP90 expression enhanced the therapeutic efficacy of an azole. Thus, harnessing Hsp90 provides a much-needed strategy for improving the treatment of fungal disease because it enhances the efficacy of existing antifungals, blocks the emergence of drug resistance, and exerts broad-spectrum activity against diverse fungal pathogens.

The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent.

ABSTRACT Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.

Inhibiting the transcription factor HSF1 as an anticancer strategy

Background: In mammals, the cytoprotective heat-shock response is regulated primarily by heat shock factor 1 (HSF1). Unfortunately, the effects of HSF1 also support the ability of cancer cells to accommodate imbalances in signaling and alterations in DNA, protein and energy metabolism associated with oncogenesis. The malignant lifestyle confers dependence on this ‘non-oncogene’, suggesting a therapeutic role for HSF1 inhibitors. Objective/methods: We begin with an overview of how HSF1 affects cancer biology and how its activity is regulated. We then summarize progress in discovery and development of HSF1 inhibitors, their current limitations and potential as anticancer agents with a fundamentally different scope of action from other clinically validated modulators of protein homeostasis. Results/conclusions: It is likely that within the next 5 years usable inhibitors of HSF1 will be identified and in early pre-clinical evaluation.

The Stress Response: Implications for the Clinical Development of Hsp90 Inhibitors

In their role as molecular chaperones, heat shock proteins serve as central integrators of protein homeostasis within cells. As part of this function, they guide the folding, assembly, intracellular disposition and proteolytic turnover of many key regulators of cell growth, differentiation and survival. Not surprisingly then, heat shock proteins are over expressed in many types of cancer, and induction of the stress response may actually be required for cells to tolerate the genetic disarray characteristic of malignant transformation. Regulation of heat shock protein levels via the stress response is complex, but recent data indicate that the molecular chaperone Hsp90 plays a key role. Specifically, Hsp90 inhibitors alter the multi-chaperone complexes associated with Heat Shock Factor 1 (HSF1), the dominant transcription factor controlling induction of the stress response, and stimulate HSF1-activated heat shock gene expression. Induction of this heat shock response has now emerged as an important consideration in the further clinical development of Hsp90 inhibitors for several reasons. First, tumors in which the stress response is compromised appear particularly sensitive to Hsp90 inhibition. Second, induction of the stress response by Hsp90 inhibitors provides a sensitive pharmacodynamic endpoint with which to monitor drug action in individual patients. Third, Hsp90 inhibitors display important therapeutic interactions with both conventional DNA-targeted chemotherapeutics and newer molecularly targeted agents. These interactions are, at least in part, due to modulation of the stress response by these drugs. Lastly, stress response induction by Hsp90 inhibitors may have therapeutic benefits in non-neoplastic disorders such as heart disease, stroke and neurodegenerative diseases. These benefits are just beginning to be explored.

High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer

Heat-shock factor 1 (HSF1) is the master transcriptional regulator of the cellular response to heat and a wide variety of other stressors. We previously reported that HSF1 promotes the survival and proliferation of malignant cells. At this time, however, the clinical and prognostic significance of HSF1 in cancer is unknown. To address this issue breast cancer samples from 1,841 participants in the Nurses’ Health Study were scored for levels of nuclear HSF1. Associations of HSF1 status with clinical parameters and survival outcomes were investigated by Kaplan–Meier analysis and Cox proportional hazard models. The associations were further delineated by Kaplan–Meier analysis using publicly available mRNA expression data. Our results show that nuclear HSF1 levels were elevated in ∼80% of in situ and invasive breast carcinomas. In invasive carcinomas, HSF1 expression was associated with high histologic grade, larger tumor size, and nodal involvement at diagnosis (P < 0.0001). By using multivariate analysis to account for the effects of covariates, high HSF1 levels were found to be independently associated with increased mortality (hazards ratio: 1.62; 95% confidence interval: 1.21–2.17; P < 0.0013). This association was seen in the estrogen receptor (ER)-positive population (hazards ratio: 2.10; 95% confidence interval: 1.45–3.03; P < 0.0001). In public expression profiling data, high HSF1 mRNA levels were also associated with an increase in ER-positive breast cancer-specific mortality. We conclude that increased HSF1 is associated with reduced breast cancer survival. The findings indicate that HSF1 should be evaluated prospectively as an independent prognostic indicator in ER-positive breast cancer. HSF1 may ultimately be a useful therapeutic target in cancer.

Tight Coordination of Protein Translation and HSF1 Activation Supports the Anabolic Malignant State

Introduction Ribosome biogenesis is commonly up-regulated to satisfy the increased anabolic demands associated with malignant transformation and tumor growth. Many different oncogenic signaling pathways converge on the ribosome to increase translational flux. Despite the detailed understanding of ribosome regulation in cancer, it is not clear whether the net translational activity of the ribosome can itself regulate transcriptional programs that support and promote the malignant state. Methods To investigate the transcriptional effects of modulating translational activity in malignant cells, we used integrated chemical and genetic approaches, including a gene signature–based genetic and chemical screen of more than 600,000 gene expression profiles (LINCS database) and an independent, reporter-based chemical screen of more than 300,000 compounds. A lead compound was tested in several cell-lines unified by their increased dependence on HSF1 activation for growth and survival, and in an in vivo cancer model. Results Inhibiting translation led to large changes in the transcriptome. The single most enriched category consisted of genes regulated by the heat-shock transcription factor, HSF1. The most down-regulated mRNA was HSPA8, which encodes the constitutive HSP70 chaperone that helps to fold nascent polypeptides. The expression of many other genes that HSF1 coordinates to support cancer were also strongly affected. HSF1 protein levels were unchanged, but HSF1 DNA occupancy was nearly eliminated. Inhibition of the HSF1-regulated gene expression program is thus a dominant transcriptional effect elicited by inhibiting protein translation. Using a gene signature of HSF1 inactivation to query the LINCS database revealed a strong connection between HSF1 inactivation and perturbations that inhibit protein translation, including a broad spectrum of chemical and genetic interventions that target the ribosome, eukaryotic initiation factors (eIFs), aminoacyl tRNA synthetases, and upstream signaling/regulatory pathways that control translation. Our high-throughput small-molecule screen identified rocaglamide A, an inhibitor of translation initiation, was the strongest inhibitor of HSF1 activation. An analog of this compound, RHT, increased thioredoxin-interacting protein (TXNIP) mRNA and protein levels and decreased glucose uptake and lactate production. Cell-based cancer models characterized by high dependence on HSF1 activation for growth and survival were highly sensitive to RHT, as were cells derived from diverse hematopoietic malignancies. RHT had a strong antitumor effect—with marked inhibition of HSF1 activity and glucose uptake—against xenografted acute myeloid leukemia cells. Discussion The ribosome functions as a central information hub in malignant cells: Translational flux conveys information about the cell’s metabolic status to regulate the transcriptional programs that support it. Multiple unbiased chemical and genetic approaches establish HSF1 as a prime transducer of this information, centrally poised to regulate the transcription of genes that support protein folding, biomass expansion, anabolic metabolism, cellular proliferation, and survival. Targeting translation initiation may offer a strategy for reversing HSF1 activation, disabling metabolic and cytoprotective pathways in malignant cells. HSF1 at the crossroads of protein translation and metabolism. (Left) Cancers activate an HSF1-regulated transcriptional program to adapt to the anabolic demands of relentless biomass expansion. Glucose uptake increases, and expression of TXNIP, an inhibitor of glucose uptake, drops. (Right) Down-regulating translation with rocaglate scaffold initiation inhibitors reverses cancer-associated HSF1 activation. Glucose uptake drops as TXNIP levels rise. Sensing Reduced Translation The interplay between metabolic pathways and the cellular survival programs that enable tumors to grow are poorly understood. Heat shock factor 1 (HSF1) coordinates an unexpectedly diverse transcriptional network involved in oncogenesis. Santagata et al. (p. 1238303; see the Perspective by Gandin and Topisirovic) found that reduced translation may be used to sense a cells metabolic status and regulate transcription, in particular by inactivating HSF1 with consequent affects on its targets. Small-molecule drugs that affected this link were able to inhibit the growth of transformed cells in culture and of an animal tumor model. Chemical and genetic screening links ribosome activity levels and a transcriptional regulator in malignant cells. [Also see Perspective by Gandin and Topisirovic] The ribosome is centrally situated to sense metabolic states, but whether its activity, in turn, coherently rewires transcriptional responses is unknown. Here, through integrated chemical-genetic analyses, we found that a dominant transcriptional effect of blocking protein translation in cancer cells was inactivation of heat shock factor 1 (HSF1), a multifaceted transcriptional regulator of the heat-shock response and many other cellular processes essential for anabolic metabolism, cellular proliferation, and tumorigenesis. These analyses linked translational flux to the regulation of HSF1 transcriptional activity and to the modulation of energy metabolism. Targeting this link with translation initiation inhibitors such as rocaglates deprived cancer cells of their energy and chaperone armamentarium and selectively impaired the proliferation of both malignant and premalignant cells with early-stage oncogenic lesions.

Geldanamycin-stimulated destabilization of mutated p53 is mediated by the proteasome in vivo.

Mutation of the tumor suppressor gene p53 is the most common genetic abnormality detected in human cancers. Wild type p53 is a short-lived protein with very low basal intracellular levels. Most mutated forms of the protein, however, display markedly increased intracellular levels as an essential feature of their positive transforming activity. In this report, we have used selective inhibitors of the 20S proteasome to demonstrate that processing of p53 by ubiquitination and proteasome-mediated degradation is impaired by commonly occuring mutations of the protein. We found that this impairment of p53 turnover can be reversed by treatment of tumor cells with the benzoquinone ansamycin, geldanamycin, leading to a marked reduction in intracellular p53 levels. Finally, using cells which over-express a mutant p53 protein, we were able to demonstrate that restoration of proteasome-mediated degradation by geldanamycin is accompanied by p53 polyubiquitination. Although much remains to be learned about the mechanisms involved, our data demonstrate that selective de-stabilization of mutant transforming proteins such as p53 can be achieved pharmacologically with agents such as geldanamycin which modify the function of molecular chaperone proteins within tumor cells.