Ramin Dubey

Inhibition of Hypoxia Inducible Factor 1–Transcription Coactivator Interaction by a Hydrogen Bond Surrogate α-Helix

Designed ligands that inhibit hypoxia-inducible gene expression could offer new tools for genomic research and, potentially, drug discovery efforts for the treatment of neovascularization in cancers. We report a stabilized alpha-helix designed to target the binding interface between the C-terminal transactivation domain (C-TAD) of hypoxia-inducible factor 1alpha (HIF-1alpha) and cysteine-histidine rich region (CH1) of transcriptional coactivator CBP/p300. The synthetic helix disrupts the structure and function of this complex, resulting in a rapid downregulation of two hypoxia-inducible genes (VEGF and GLUT1) in cell culture.

Direct inhibition of hypoxia-inducible transcription factor complex with designed dimeric epidithiodiketopiperazine.

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling

Significance Protein–protein interactions are attractive targets for interfering with processes leading to disease states. Proteins often use folded domains or secondary structures to contact partner proteins. Synthetic molecules that mimic these domains could disrupt protein–protein contacts, thereby inhibiting formation of multiprotein complexes. This article describes protein domain mimetics (PDMs) that modulate interactions between two proteins that control expression of a multitude of genes under hypoxic environments, such as those found inside tumors. The low-oxygen conditions promote angiogenesis—process of formation of new blood vessels—that together with invasion and altered energy metabolism facilitates tumor growth. We find that the PDMs can control expression of target hypoxia-inducible genes in cell culture and reduce tumor burden in mice. Selective blockade of gene expression by designed small molecules is a fundamental challenge at the interface of chemistry, biology, and medicine. Transcription factors have been among the most elusive targets in genetics and drug discovery, but the fields of chemical biology and genetics have evolved to a point where this task can be addressed. Herein we report the design, synthesis, and in vivo efficacy evaluation of a protein domain mimetic targeting the interaction of the p300/CBP coactivator with the transcription factor hypoxia-inducible factor-1α. Our results indicate that disrupting this interaction results in a rapid down-regulation of hypoxia-inducible genes critical for cancer progression. The observed effects were compound-specific and dose-dependent. Gene expression profiling with oligonucleotide microarrays revealed effective inhibition of hypoxia-inducible genes with relatively minimal perturbation of nontargeted signaling pathways. We observed remarkable efficacy of the compound HBS 1 in suppressing tumor growth in the fully established murine xenograft models of renal cell carcinoma of the clear cell type. Our results suggest that rationally designed synthetic mimics of protein subdomains that target the transcription factor–coactivator interfaces represent a unique approach for in vivo modulation of oncogenic signaling and arresting tumor growth.

Suppression of tumor growth by designed dimeric epidithiodiketopiperazine targeting hypoxia-inducible transcription factor complex.

Hypoxia is a hallmark of solid tumors, is associated with local invasion, metastatic spread, resistance to chemo- and radiotherapy, and is an independent, negative prognostic factor for a diverse range of malignant neoplasms. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, altered energy metabolism, antiapoptotic, and pro-proliferative mechanisms that promote tumor growth. A designed transcriptional antagonist, dimeric epidithiodiketopiperazine (ETP 2), selectively disrupts the interaction of HIF1α with p300/CBP coactivators and downregulates the expression of hypoxia-inducible genes. ETP 2 was synthesized via a novel homo-oxidative coupling of the aliphatic primary carbons of the dithioacetal precursor. It effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of cell lines representing breast and lung cancers. We observed an outstanding antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. Treatment with either form of ETP 2 (1 mg/kg) resulted in a rapid regression of tumor growth that lasted for up to 14 days. These results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

Significance Cell-based approaches utilizing retinal pigment epithelial (RPE)-like cells derived from human pluripotent stem cells (hPSCs) are being developed for the treatment of retinal degeneration. In most research published to date, the choice of the factors used to induce RPE differentiation is based on data from developmental studies. Here, we developed an unbiased approach directed at identifying novel RPE differentiation-promoting factors using a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. We identified chetomin, a dimeric epidithiodiketopiperazine, as a strong inducer of RPE; combination with nicotinamide resulted in efficient RPE differentiation. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems. DOI: http://dx.doi.org/10.7554/eLife.21459.001

Chromatin-Remodeling Complex SWI/SNF Controls Multidrug Resistance by Transcriptionally Regulating the Drug Efflux Pump ABCB1.

Anthracyclines are among the most effective yet most toxic drugs used in the oncology clinic. The nucleosome-remodeling SWI/SNF complex, a potent tumor suppressor, is thought to promote sensitivity to anthracyclines by recruiting topoisomerase IIa (TOP2A) to DNA and increasing double-strand breaks. In this study, we discovered a novel mechanism through which SWI/SNF influences resistance to the widely used anthracycline doxorubicin based on the use of a forward genetic screen in haploid human cells, followed by a rigorous single and double-mutant epistasis analysis using CRISPR/Cas9-mediated engineering. Doxorubicin resistance conferred by loss of the SMARCB1 subunit of the SWI/SNF complex was caused by transcriptional upregulation of a single gene, encoding the multidrug resistance pump ABCB1. Remarkably, both ABCB1 upregulation and doxorubicin resistance caused by SMARCB1 loss were dependent on the function of SMARCA4, a catalytic subunit of the SWI/SNF complex. We propose that residual SWI/SNF complexes lacking SMARCB1 are vital determinants of drug sensitivity, not just to TOP2A-targeted agents, but to the much broader range of cancer drugs effluxed by ABCB1. Cancer Res; 76(19); 5810-21. ©2016 AACR.

Tumor targeting, trifunctional dendritic wedge.

We report in vitro and in vivo evaluation of a newly designed trifunctional theranostic agent for targeting solid tumors. This agent combines a dendritic wedge with high boron content for boron neutron capture therapy or boron MRI, a monomethine cyanine dye for visible-light fluorescent imaging, and an integrin ligand for efficient tumor targeting. We report photophysical properties of the new agent, its cellular uptake and in vitro targeting properties. Using live animal imaging and intravital microscopy (IVM) techniques, we observed a rapid accumulation of the agent and its retention for a prolonged period of time (up to 7 days) in fully established animal models of human melanoma and murine mammary adenocarcinoma. This macromolecular theranostic agent can be used for targeted delivery of high boron load into solid tumors for future applications in boron neutron capture therapy.

Abstract PR05: Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling

Wnt signaling is at the core of animal development and regeneration. It controls cell proliferation, differentiation and survival. In humans, defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer (CRC). A convergence of methodological advances enabled us to do a systematic forward genetic analysis of canonical Wnt signaling in human cells, revealing new players and regulatory mechanisms. Using HAP1 cells, a haploid human cell line, we conducted a set of genome-wide screens to interrogate the Wnt pathway under normal and pathological conditions. We constructed a HAP1 line harboring a Wnt-responsive GFP reporter, mutagenized the cells by insertion of a gene trap retrovirus throughout the haploid genome, and sorted for cells exhibiting phenotypes of interest using FACS. By setting appropriate gates we enriched for negative and positive regulators of the pathway. These screens yielded many known players as well as a new transcription factor, TFAP4, required for signaling downstream of β-catenin. Unexpectedly AXIN2, a scaffold in the β-catenin destruction complex, was a prominent hit in the screen for positive regulators. However, the distribution of gene trap insertions in AXIN2 suggested that truncation of the last one or two exons encompassing the DAX domain, as opposed to disruption of the entire AXIN2 gene, was responsible for the observed phenotype. Follow-up analysis of cells in which AXIN1 was eliminated and AXIN2 C-terminal truncations were generated at the single endogenous locus confirmed that this domain is dispensable for destruction complex function. Furthermore, in these cells responsiveness to Wnt was only partially diminished, suggesting that the DAX domain is not essential for transduction of the Wnt signal from the receptor. Instead we show that the C-terminus is involved in regulating Axin2 protein levels. Using CRISPR/CAS9 we then generated haploid cell lines lacking adenomatous polyposis coli (APC), a scaffold in the β-catenin destruction complex, or casein kinase α (CK1α), the priming kinase for β-catenin degradation. These lines have constitutive Wnt signaling activity, and the former recapitulates the APC mutations found in CRC patients. We conducted synthetic genome-wide screens looking for modifiers of these mutations in an otherwise isogenic background. We found that in APC-null cells, deletion of the RNA binding protein SERBP1 reduces β-catenin protein levels significantly, restoring normal levels of Wnt-stimulated signaling. In CK1α-null cells, deletion of the E3 ubiquitin ligase HUWE1 decreases Wnt signaling by over 85% with only a minor reduction of β-catenin protein level. This defect is specific to the CK1α genetic background, since deletion of HUWE1 in APC-null cells has no effect on signaling. These results suggest a destruction complex independent role of CK1α in Wnt signaling, mediated by HUWE1. Our findings are supported by experiments in model organisms. The experimental approaches used in this study are generally applicable to other signaling pathways and more broadly to any cellular process in which a phenotypic readout can be used to enrich for mutant cells. The combination of forward genetics in haploid cells and CRISPR/CAS9-based genome engineering brings to bear on cultured human cells the immense power of genetics traditionally limited to model organisms such as yeast. This abstract is also presented as Poster B11. Citation Format: Andres M. Lebensohn, Ramin Dubey, Leif R. Neitzel, Ofelia Tacchelly, Caleb D. Marceau, Zahra Bahrami, Amanda G. Hansen, Yashi Ahmed, Ethan Lee, Jan Carette, Rajat Rohatgi. Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR05.

Abstract 1014: Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Traditional anticancer therapies are limited in their efficacy because they often lead to drug-resistant tumors and display toxicity to normal cells. Control of angiogenesis represents one of the key goals in cancer therapy, as exemplified by targeting the VEGF pathway with bevacizumab.Chronic hypoxia, a hallmark of many solid tumors is responsible for the elevated levels of VEGF and other pro-proliferative factors. The cellular response to hypoxia is primarily mediated by a family of transcription factors, among which hypoxia-inducible factor 1 (HIF1) plays a major role. Under normoxia, the oxygen-sensitive α subunit of HIF1 is rapidly and constitutively degraded but is stabilized and accumulates under hypoxia. Upon nuclear translocation, HIF1 controls the expression of over 100 genes involved in angiogenesis, and pro-proliferative mechanisms that promote tumor growth. The specific hypothesis behind the proposed research is that angiogenesis could be effectively controlled by down-regulation of hypoxia-inducible gene expression via a disruption of key transcription factor-coactivator interactions with designed molecules. The team at BioCycive and its collaborators at USC are currently developing such compounds that block hypoxia-inducible transcription of key genes. We have designed and synthesized a novel dimeric epidithiodiketopiperazine, ETP2, which targets pleiotropic coactivator p300 disrupts HIF1α-p300 complex in vitro and effectively down-regulates hypoxia-inducible signaling and gene expression. ETP 2 effectively inhibits HIF1-induced activation of VEGFA, LOX, Glut1, and c-Met genes in a panel of breast and lung cancer cell lines. We have shown excellent antitumor efficacy of both (±)-ETP 2 and meso-ETP 2 in a fully established breast carcinoma model by intravital microscopy. A significant decrease in the vascularization of these tumors upon treatment with ETP 2 was also noticed, which is consistent with the proposed mechanism of inhibition of vascularization via down-regulation of hypoxia-inducible signaling.Our efficacy studies with renal carcinoma cells lines resulted in similar findings, where we successfully reached the primary endpoint of efficacy (reduction in tumor volume of >50%) and secondary endpoint of survival. Nude mice engrafted with 786-O (renal cell carcinoma) were treated with ETPs at 1 mg/kg given IV every three days for 45 days. All tumors were imaged at the end of the study and harvested for histopathology studies. MTD studies clearly indicated that ETP 2 was non-toxic within the range of injected doses, with an average MTD of 21.1 mg/kg. Our preliminary PK studies show that the ETP 2 is a lipophilic molecule, with a plasma half-life of 3.5-5 days. Our results suggest that inhibition of HIF1 transcriptional activity by designed dimeric ETPs could offer an innovative approach to cancer therapy with the potential to overcome hypoxia-induced tumor growth and resistance. Citation Format: Ramin Dubey, Ivan Grishagin, Usha Nagavarapu, Chenera Balan, Shalabh Gupta, Bogdan Z. Olenyuk. Novel selective HIF1 alpha inhibitor: Well tolerated with excellent efficacy in renal cell cancer xenograft studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2014-1014