Faculty Bibliography

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12^F36Vwith expression of FKBP12^F36Vin-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRAS^G12Vloss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

Small cell lung cancer (SCLC) patient-derived xenografts (PDXs) can be generated from biopsies or circulating tumor cells (CTCs), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC-enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a two-year timeframe, at 89% and 38% efficiency, respectively. Whole exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and cisplatin (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a Kras^G12D/+ mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx, Ptip, Acp5, Acacb, and Clu, whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the Kras^G12D/+ mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx-knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis.

Tuberous sclerosis complex (TSC) is an incurable multisystem disease characterized by mTORC1-hyperactive tumors. TSC1/2 mutations also occur in other neoplastic disorders, including lymphangioleiomyomatosis (LAM) and bladder cancer. Whether TSC-associated tumors will respond to immunotherapy is unknown. We report here that the programmed death 1 coinhibitory receptor (PD-1) is upregulated on T cells in renal angiomyolipomas (AML) and pulmonary lymphangioleiomyomatosis (LAM). In C57BL/6J mice injected with syngeneic TSC2-deficient cells, anti-PD-1 alone decreased 105K tumor growth by 67% (P < 0.0001); the combination of PD-1 and CTLA-4 blockade was even more effective in suppressing tumor growth. Anti-PD-1 induced complete rejection of TSC2-deficient 105K tumors in 37% of mice (P < 0.05). Double blockade of PD-1 and CTLA-4 induced rejection in 62% of mice (P < 0.01). TSC2 reexpression in TSC2-deficient TMKOC cells enhanced antitumor immunity by increasing T cell infiltration and production of IFN-γ/TNF-α by T cells, suggesting that TSC2 and mTORC1 play specific roles in the induction of antitumor immunity. Finally, 1 month of anti-PD-1 blockade reduced renal tumor burden by 53% (P < 0.01) in genetically engineered Tsc2+/- mice. Taken together, these data demonstrate for the first time to our knowledge that checkpoint blockade may have clinical efficacy for TSC and LAM, and possibly other benign tumor syndromes, potentially yielding complete and durable clinical responses.

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

BACKGROUND:Reduced leukocyte telomere length (LTL) has been found to be associated with multiple common age-related diseases, including heart disease, diabetes, and cancer. A link has also been suggested between shortened LTL and major depressive disorder (MDD), suggesting that MDD may be a disease of accelerated aging. This prospective, longitudinal study examined the association between depression diagnosis at baseline and change in LTL over two years in a well-characterized sample of N = 117 adults with or without MDD at baseline, using rigorous entry criteria. METHODS:Participants aged 18-70 were assessed with validated instruments by trained, doctoral-level clinician raters at baseline and at two-year follow-up, and blood samples were obtained at both visits. LTL was assayed under identical methods using quantitative polymerase chain reaction (qPCR). The effect of an MDD diagnosis at baseline on change in LTL over two years was examined via hierarchical mixed models, which included potential confounders. RESULTS:Individuals with MDD at baseline had greater LTL shortening over two years than individuals without MDD (p = 0.03), even after controlling for differences in age, sex, and body mass index (BMI). In the sub-sample of individuals with MDD diagnoses at baseline, no significant associations between LTL change and symptom severity or duration were found. CONCLUSION/CONCLUSIONS:A baseline diagnosis of MDD prospectively predicted LTL shortening over two years. Our results provide further support for MDD as a disease associated with accelerated aging in a well-characterized sample using validated, clinician-rated measures.

Autophagy has been shown to be elevated in pancreatic adenocarcinoma (PDAC) and its role in promoting established tumor growth has made it a promising therapeutic target. However, due to limitations of prior mouse models as well as the lack of potent and selective autophagy inhibitors, the ability to fully assess the mechanistic basis of how autophagy supports pancreatic cancer has been limited. To test the feasibility of treating PDAC using autophagy inhibition and further our understanding of the mechanisms of pro-tumor effects of autophagy, we developed a novel mouse model that allowed the acute and reversible inhibition of autophagy. We observed that autophagy inhibition causes significant tumor regression in an autochthonous mouse model of PDAC. A detailed analysis of these effects indicated that the tumor regression was likely multifactorial, involving both tumor cell intrinsic as well as host effects. Thus, our study supports autophagy inhibition in PDAC may have future utility in the treatment of pancreatic cancer and illustrates the importance of assessing complex biological processes in relevant autochthonous models.

An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells which survive despite effective eradication of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy,a combination previously shown to suppress drug tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response (UPR) associated with STING upregulation, promoting pro-tumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications.