Faculty Bibliography

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apoB48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cell expression systems are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The cell culture system was developed by ablating the endogenous MTTP gene in human hepatoma Huh-7 cells using sgRNA and CRISPR/Cas9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA, or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins, and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer with limited meaningful treatment options. NEPC lesions uniquely express delta-like ligand 3 (DLL3) on their cell surface. Taking advantage of DLL3 overexpression, we developed and evaluated lutetium-177 (¹⁷⁷Lu)-labeled DLL3-targeting antibody SC16 (¹⁷⁷Lu-DTPA-SC16) as a treatment for NEPC. SC16 was functionalized with DTPA-CHX-A" chelator and radiolabeled with ¹⁷⁷Lu to produce ¹⁷⁷Lu-DTPA-SC16. Specificity and selectivity of ¹⁷⁷Lu-DTPA-SC16 were evaluated in vitro and in vivo using NCI-H660 (NEPC, DLL3-positive) and DU145 (adenocarcinoma, DLL3-negative) cells and xenografts. Dose-dependent treatment efficacy and specificity of ¹⁷⁷Lu-DTPA-SC16 radionuclide therapy were evaluated in H660 and DU145 xenograft-bearing mice. Safety of the agent was assessed by monitoring hematologic parameters. ¹⁷⁷Lu-DTPA-SC16 showed high tumor uptake and specificity in H660 xenografts, with minimal uptake in DU145 xenografts. At all three tested doses of ¹⁷⁷Lu-DTPA-SC16 (4.63, 9.25, and 27.75 MBq/mouse), complete responses were observed in H660-bearing mice; 9.25 and 27.75 MBq/mouse doses were curative. Even the lowest tested dose proved curative in five (63%) of eight mice, and recurring tumors could be successfully re-treated at the same dose to achieve complete responses. In DU145 xenografts, ¹⁷⁷Lu-DTPA-SC16 therapy did not inhibit tumor growth. Platelets and hematocrit transiently dropped, reaching nadir at 2 to 3 wk. This was out of range only in the highest-dose cohort and quickly recovered to normal range by week 4. Weight loss was observed only in the highest-dose cohort. Therefore, our data demonstrate that ¹⁷⁷Lu-DTPA-SC16 is a potent and safe radioimmunotherapeutic agent for testing in humans with NEPC.

Access to clinically relevant small cell lung cancer (SCLC) tissue is limited because surgical resection is rare in metastatic SCLC. Patient-derived xenografts (PDX) and circulating tumor cell-derived xenografts (CDX) have emerged as valuable tools to characterize SCLC. Here, we present a resource of 46 extensively annotated PDX/CDX models derived from 33 patients with SCLC. We perform multi-omic analyses, using targeted tumor next-generation sequencing, RNA-sequencing, and immunohistochemistry to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these SCLC models. SCLC subtypes characterized by transcriptional regulators, ASCL1, NEUROD1 and POU2F3 are confirmed in this cohort. A subset of SCLC clinical specimens, including matched PDX/CDX and clinical specimen pairs, confirm that the primary features and genomic and proteomic landscapes of the tumors of origin are preserved in the derivative PDX models. This resource provides a powerful system to study SCLC biology.

PURPOSE/OBJECTIVE:Small cell lung cancer is an exceptionally lethal form of lung cancer with limited treatment options. Delta-like ligand 3 (DLL3) is an attractive therapeutic target as surface expression is almost exclusive to tumor cells. EXPERIMENTAL DESIGN/METHODS:Lu]Lu-DTPA-CHX-A"-SC16 binds to DLL3 on SCLC cells and delivers targeted radiotherapy while minimizing radiation to healthy tissue. RESULTS:Lu]Lu-DTPA-CHX-A"-SC16.

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease.

Lineage plasticity is implicated in treatment resistance in multiple cancers. In lung adenocarcinomas (LUADs) amenable to targeted therapy, transformation to small cell lung cancer (SCLC) is a recognized resistance mechanism. Defining molecular mechanisms of neuroendocrine (NE) transformation in lung cancer has been limited by a paucity of pre-/post-transformation clinical samples. Detailed genomic, epigenomic, transcriptomic, and protein characterization of combined LUAD/SCLC tumors, as well as pre-/post-transformation samples, support that NE transformation is primarily driven by transcriptional reprogramming rather than mutational events. We identify genomic contexts in which NE transformation is favored, including frequent loss of the 3p chromosome arm. We observed enhanced expression of genes involved in PRC2 complex and PI3K/AKT and NOTCH pathways. Pharmacological inhibition of the PI3K/AKT pathway delayed tumor growth and NE transformation in an EGFR-mutant patient-derived xenograft model. Our findings define a novel landscape of potential drivers and therapeutic vulnerabilities of neuroendocrine transformation in lung cancer.

Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf-Mycl-driven mouse model of SCLC. Rlf-Mycl fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the Rlf-Mycl genetically engineered mouse model displayed gene expression similarities with human Rlf-Mycl SCLC. Together, our studies support Rlf-Mycl as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. SIGNIFICANCE: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame Rlf-Mycl gene fusion by developing a Rlf-Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis.

Activation of mitogenic signaling pathways is a common oncogenic driver of many solid tumors including lung cancer. Although activating mutations in the mitogen-activated protein kinase (MAPK) pathway are prevalent in non-small cell lung cancers, MAPK pathway activity, counterintuitively, is relatively suppressed in the more aggressively proliferative small cell lung cancer (SCLC). Here, we elucidate the role of the MAPK pathway and how it interacts with other signaling pathways in SCLC. We find that the most common SCLC subtype, SCLC-A associated with high expression of ASCL1, is selectively sensitive to MAPK activation in vitro and in vivo through induction of cell-cycle arrest and senescence. We show strong upregulation of ERK negative feedback regulators and STAT signaling upon MAPK activation in SCLC-A lines. These findings provide insight into the complexity of signaling networks in SCLC and suggest subtype-specific mitogenic vulnerabilities.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation.