Chun-Hao Huang

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)–dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma

One-year survival rates for newly diagnosed hepatocellular carcinoma (HCC) are <50%, and unresectable HCC carries a dismal prognosis owing to its aggressiveness and the undruggable nature of its main genetic drivers. By screening a custom library of shRNAs directed toward known drug targets in a genetically defined Myc-driven HCC model, we identified cyclin-dependent kinase 9 (Cdk9) as required for disease maintenance. Pharmacological or shRNA-mediated CDK9 inhibition led to robust anti-tumor effects that correlated with MYC expression levels and depended on the role that both CDK9 and MYC exert in transcription elongation. Our results establish CDK9 inhibition as a therapeutic strategy for MYC-overexpressing liver tumors and highlight the relevance of transcription elongation in the addiction of cancer cells to MYC.

BRD4 Connects Enhancer Remodeling to Senescence Immune Surveillance

UNLABELLED Oncogene-induced senescence is a potent barrier to tumorigenesis that limits cellular expansion following certain oncogenic events. Senescent cells display a repressive chromatin configuration thought to stably silence proliferation-promoting genes while simultaneously activating an unusual form of immune surveillance involving a secretory program referred to as the senescence-associated secretory phenotype (SASP). Here, we demonstrate that senescence also involves a global remodeling of the enhancer landscape with recruitment of the chromatin reader BRD4 to newly activated super-enhancers adjacent to key SASP genes. Transcriptional profiling and functional studies indicate that BRD4 is required for the SASP and downstream paracrine signaling. Consequently, BRD4 inhibition disrupts immune cell-mediated targeting and elimination of premalignant senescent cells in vitro and in vivo Our results identify a critical role for BRD4-bound super-enhancers in senescence immune surveillance and in the proper execution of a tumor-suppressive program. SIGNIFICANCE This study reveals how cells undergoing oncogene-induced senescence acquire a distinctive enhancer landscape that includes formation of super-enhancers adjacent to immune-modulatory genes required for paracrine immune activation. This process links BRD4 and super-enhancers to a tumor-suppressive immune surveillance program that can be disrupted by small molecule inhibitors of the bromo and extra terminal domain family of proteins. Cancer Discov; 6(6); 612-29. ©2016 AACR.See related commentary by Vizioli and Adams, p. 576This article is highlighted in the In This Issue feature, p. 561.

Prediction of potent shRNAs with a sequential classification algorithm

We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel data sets, SplashRNA outperforms previous algorithms and reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration. SplashRNA can significantly improve the accuracy of loss-of-function genetics studies and facilitates the generation of compact shRNA libraries.

Chelator-Free Radiolabeling of SERRS Nanoparticles for Whole-Body PET and Intraoperative Raman Imaging

A single contrast agent that offers whole-body non-invasive imaging along with the superior sensitivity and spatial resolution of surface-enhanced resonance Raman scattering (SERRS) imaging would allow both pre-operative mapping and intraoperative imaging and thus be highly desirable. We hypothesized that labeling our recently reported ultrabright SERRS nanoparticles with a suitable radiotracer would enable pre-operative identification of regions of interest with whole body imaging that can be rapidly corroborated with a Raman imaging device or handheld Raman scanner in order to provide high precision guidance during surgical procedures. Here we present a straightforward new method that produces radiolabeled SERRS nanoparticles for combined positron emission tomography (PET)-SERRS tumor imaging without requiring the attachment of molecular chelators. We demonstrate the utility of these PET-SERRS nanoparticles in several proof-of-concept studies including lymph node (LN) tracking, intraoperative guidance for LN resection, and cancer imaging after intravenous injection. We anticipate that the radiolabeling method presented herein can be applied generally to nanoparticle substrates of various materials by first coating them with a silica shell and then applying the chelator-free protocol.

Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis

Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1753-62. ©2017 AACR.

Abstract LB-056: TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer

Castration-resistant prostate cancer (CRPC) is one of the most difficult cancers to treat with conventional methods and is responsible for nearly all prostate cancer deaths in the US. The Sawyers laboratory first showed that the primary mechanism of resistance to antiandrogen therapy is elevated androgen receptor (AR) expression. Research based on this finding has led to the development of next-generation antiandrogen: enzalutamide. Despite the exciting clinical success of enzalutamide, about 60% of patients exhibit various degrees of resistance to this agent. Highly variable responses to enzalutamide limit the clinical benefit of this novel antiandrogen, underscoring the importance of understanding the mechanisms of enzalutamide resistance. Most recently, an unbiased SU2C-Prostate Cancer Dream Team metastatic CRPC sequencing project led by Dr. Sawyers and Dr. Chinnaiyan revealed that mutations in the TP53 locus are the most significantly enriched alteration in CRPC tumors when compared to primary prostate cancers. Moreover, deletions and decreased expressions of the TP53 and RB1 loci (co-occurrence and individual occurrence) are more commonly associated with CRPC than with primary tumors. These results established that alteration of the TP53 and RB1 pathways are associated with the development of antiandrogen resistance. By knockdowning TP53 or/and RB1 in the castration resistant LNCaP/AR model, we demonstrate that the disruption of either TP53 or RB1 alone confers significant resistance to enzalutamide both in vitro and in vivo. Strikingly, the co-inactivation of these pathways confers the most dramatic resistance. Since up-regulation of either AR or AR target genes is not observed in the resistant tumors, loss of TP53 and RB1 function confers enzalutamide resistance likely through an AR independent mechanism. In the clinic, resistance to enzalutamide is increasingly being associated with a transition to a poorly differentiated or neuroendocrine-like histology. Interestingly, we observed significant up-regulations of the basal cell marker Ck5 and the neuroendocrine-like cell marker Synaptophysin in the TP53 and RB1 inactivated cells, as well as down-regulation of the luminal cell marker Ck8. The differences between these markers became even greater after enzalutamide treatment. By using the p53-stabilizing drug Nutlin, level of p53 is rescued and consequently the the decrease of AR protein caused by RB1 and TP53 knockdown is reversed. These results strongly suggest that interference of TP53 and RB1 pathways confers antiandrogen resistance by “priming” prostate cancer cells to reprogramming or transdifferentiation, likely neuroendocrine-like differentiation, in response to treatment. Futher experiments will be performed to assess the molecular mechanism of TP53/RB1 alterations in mediating cell programming and conferring antiandrogen resistance. Citation Format: Ping Mu, Zhen Cao, Elizabeth Hoover, John Wongvipat, Chun-Hao Huang, Wouter Karthaus, Wassim Abida, Elisa De Stanchina, Charles Sawyers. TP53 and RB1 alterations promote reprogramming and antiandrogen resistance in advanced prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-056. doi:10.1158/1538-7445.AM2015-LB-056

Abstract 2694: Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis

Chromosomal rearrangements of the Mixed-Lineage Leukemia (MLL) gene are found in 5-10% of all patients with acute leukemia and associated with a poor prognosis. MLL-rearrangements are more frequently present in pediatric and infant patients where AF9 is one of the most common fusion partners. In order to identify novel druggable targets in MLL-AF9 rearranged leukemia, we conducted a chromatin regulator focused RNAi screen in murine MLL-AF9 leukemia cells and found hairpins targeting (K)Lysine Acetyltransferase 8 (Kat8, also known as Mof) and the previously identified target Bromodomain Containing 4 (Brd4), to be the most potent suppressors of cell growth. MOF is a histone 4 lysine 16 (H4K16) acetyltransferase and member of the MYST family of histone acetyltransferases (HATs). MOF has been shown to be crucial for murine embryogenesis and is a cell-type dependent regulator of chromatin state and various cellular processes such as T-cell differentiation, DNA damage response and cell cycle progression. Using a conditional murine Mof knockout system, we studied the role of MOF in MLL-AF9 leukemogenesis in detail. In vitro inactivation of Mof in MLL-AF9 transformed mouse hematopoietic stem and progenitor cells led to impaired colony-forming capacity. The specificity of this phenotype was shown by expression of exogenous full-length Mof, which fully rescued transformed cells from the dramatic phenotype. Inactivation of Mof in vivo, lead to reduced tumor burden and prolonged survival of mice bearing MLL-AF9 leukemia cells. RNA sequencing data comparing MLL-AF9 cells with homozygous Mof loss to a wild type control, showed a significant enrichment of genes within the apoptosis (NES 1.98, FDR-q These results indicate that MOF HAT activity is required for MLL-AF9 leukemia maintenance. Our data further suggest that MOF HAT activity may be a good target for new small molecule inhibitor development for the treatment of patients with MLL-AF9 rearranged leukemia. Citation Format: Daria G. Valerio, Haiming Xu, Chun-Wei Chen, Takayuki Hoshii, Meghan Eisold, Christopher Delaney, Monica Cusan, Aniruddha J. Deshpande, Chun-Hao Huang, Amaia Lujambio, George Zheng, Tej K. Pandita, Scott W. Lowe, Scott A. Armstrong. Histone acetyltransferase activity of MOF is required for MLL-AF9 leukemogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2694.

Abstract 2935: RNAi screen identifies therapeutic targets in hepatocellular carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Liver cancer is the third leading cause of cancer related mortality worldwide. Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a very aggressive type of cancer that lacks effective treatment, in part, due to the undruggable nature of its main genetic drivers, such as oncogene MYC amplification or loss of tumor suppressor TP53. The introduction of the multikinase inhibitor Sorafenib represents the biggest therapeutic advance in the past decade, though its activity only increases the life expectancy of liver cancer patients from 8 to 11 months. To identify new therapeutic targets for HCC, we employ RNA interference (RNAi) screens as a non-biased means to identify genes required for tumor maintenance in genetically defined tumors. In the current study, we applied an optimized shRNA platform to perform a negative selection screen in genetically-defined murine hepatocellular carcinoma cell in vitro. Specifically, we have designed a library that contains 2,245 shRNAs targeting 442 “drugged” genes, including genes encoding proteins targeted by FDA-approved drugs, small molecules in clinical trials, or compounds under preclinical development. Countersceening and validation was performed in normal murine cells and human HCC cell lines and xenograft using shRNAs and corresponding small molecular drugs. Pharmacological or shRNA-mediated inhibition of certain targets led to robust antitumor effects, both in vitro and in vivo, and suggest new strategies to inhibit Myc function therapeutically. Our results highlight the utility of RNAi screening for studying cancer vulnerabilities that can be exploited for direct pharmacological intervention. Citation Format: Chun-Hao Huang, Amaia Lujambio, Johannes Zuber, Thomas Kitzing, Darjus F. Tschaharganeh, Elisa De Stanchina, Scott W. Lowe. RNAi screen identifies therapeutic targets in hepatocellular carcinoma. [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 2935. doi:10.1158/1538-7445.AM2014-2935