Faculty Bibliography

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.

Treatment-induced neuroendocrine prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (CRPC). Using the zirconium-89 (⁸⁹Zr)-labeled DLL3 targeting antibody SC16 (⁸⁹Zr-DFO-SC16), we have developed a positron emission tomography (PET) agent to non-invasively identify the presence of DLL3-positive NEPC lesions. Methods: qPCR and immunohistochemistry were used to compare relative levels of androgen receptor (AR)-regulated markers and NEPC marker DLL3 in a panel of prostate cancer cell lines. PET imaging with ⁸⁹Zr-DFO-SC16, ⁶⁸Ga-PSMA-11, and ⁶⁸Ga-DOTA-TATE was performed in H660 NEPC xenografted male nude mice. ⁸⁹Zr-DFO-SC16 uptake was corroborated by biodistribution studies. Results: In vitro studies demonstrate H660 are positive for DLL3 and negative for AR, prostate-specific antigen (PSA), and prostate-specific membrane antigen (PSMA) both at the transcriptional and translational levels. PET imaging and biodistribution studies confirm ⁸⁹Zr-DFO-SC16 uptake is restricted to H660 tumor xenografts with background uptake in non-NEPC lesions (both AR-dependent and AR-independent). Conversely, H660 xenografts cannot be detected with imaging agents targeting PSMA (⁶⁸Ga-PSMA-11) or somatostatin receptor subtype 2 (SSTR2) (⁶⁸Ga-DOTA-TATE). Conclusion: These studies demonstrate H660 NEPC cells selectively express DLL3 on their cell surface and can be non-invasively identified with ⁸⁹Zr-DFO-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.

Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease comprising 13% of all lung cancer cases. With limited treatment options that typically result in transient responses, SCLC is responsible for approximately 250,000 deaths globally per year. The recent addition of immunotherapy to first-line platinum-based doublet chemotherapy shows only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second-line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need.
Method(s): To identify targets sensitizing to chemotherapy we performed an in vitro CRISPR screen in SCLC cell lines from all major SCLC subtypes. Candidate hits were validated genetically, and pharmacologically with in vitro synergy assays and PDX treatments. Signaling pathways were studied by western blot, and toxicity studies were performed in vivo, to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs).
Result(s): Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a contributor to chemotherapy resistance in all SCLC subtypes. Combination of selinexor, an Exportin 1 inhibitor approved for clinical use in hematological malignancies, with cisplatin or irinotecan demonstrated synergy in vitro and exquisite efficacy in vivo in chemonaive and chemoresistant SCLC PDXs representing all SCLC subtypes. This efficacy was associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation. The combinations were well tolerated in mice. We found SCLC to have the highest XPO1 mRNA expression among a diverse array of tumor histologies, which was confirmed at the protein level in clinical TMAs.
Conclusion(s): Exportin 1 inhibition enhances sensitivity to the chemotherapeutic drugs used in first-line and second-line treatment of SCLC tumors. Our results provide preclinical rationale for the combination of selinexor with cisplatin or irinotecan in naive and relapsed SCLC, respectively. Legal entity responsible for the study: The authors.
Funding(s): Supported by NCI R01 CA197936 and U24 CA213274 (CMR), the Druckenmiller Center for Lung Cancer Research (CMR, TS, AQV), NIH K08 CA-248723 (AC) and the Van Andel Institute - Stand Up to Cancer Epigenetics Dream Team grant (CMR). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. We acknowledge the use of the Integrated Genomics Operation Core, funded by the NCI Cancer Center Support Grant (CCSG, P30 CA08748), Cycle for Survival, and the Marie-Josee and Henry R. Kravis Center for Molecular Oncology. The Precision Pathology Center is supported by the NCI Cancer Center Support Grant P30-CA008748. Disclosure: A. Quintanal-Villalonga: Financial Interests, Personal, Invited Speaker: AstraZeneca. M. Offin: Financial Interests, Personal, Other: PharmaMar; Financial Interests, Personal, Other: Novartis; Financial Interests, Personal, Other: Targeted Oncology; Financial Interests, Personal, Other: Bristol Myers Squib; Financial Interests, Personal, Other: Merck Sharp & Dohme. C.M. Rudin: Financial Interests, Personal, Other: AbbVie; Financial Interests, Personal, Other: Amgen; Financial Interests, Personal, Other: Ascentage; Financial Interests, Personal, Other: AstraZeneca; Financial Interests, Personal, Other: Bicycle; Financial Interests, Personal, Other: Celgene; Financial Interests, Personal, Other: Daiichi Sankyo; Financial Interests, Personal, Other: Genentech/Roche; Financial Interests, Personal, Other: Ipsen; Financial Interests, Personal, Other: Jazz; Financial Interests, Personal, Other: Lilly; Financial Interests, Personal, Other: Pfizer; Financial Interests, Personal, Other: PharmaMar; Financial Interests, Personal, Other: Syros; Financial Interests, Personal, Other: Vavotek; Financial Interests, Personal, Advisory Board: Bridge Medicines; Financial Interests, Personal, Advisory Board: Earli; Financial Interests, Personal, Advisory Board: Harpoon Therapeutics. All other authors have declared no conflicts of interest.
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Delta-like ligand 3 (DLL3) is a therapeutic target for the treatment of small cell lung cancer, neuroendocrine prostate cancer, and isocitrate dehydrogenase mutant glioma. In the clinic, DLL3-targeted ⁸⁹Zr-immunoPET has the potential to aid in the assessment of disease burden and facilitate the selection of patients suitable for therapies that target the antigen. The overwhelming majority of ⁸⁹Zr-labeled radioimmunoconjugates are synthesized via the random conjugation of desferrioxamine (DFO) to lysine residues within the immunoglobulin. While this approach is admittedly facile, it can produce heterogeneous constructs with suboptimal in vitro and in vivo behavior. In an effort to circumvent these issues, we report the development and preclinical evaluation of site-specifically labeled radioimmunoconjugates for DLL3-targeted immunoPET. To this end, we modified a cysteine-engineered variant of the DLL3-targeting antibody SC16-MB1 with two thiol-reactive variants of DFO: one bearing a maleimide moiety (Mal-DFO) and the other containing a phenyloxadiazolyl methyl sulfone group (PODS-DFO). In an effort to obtain immunoconjugates with a DFO-to-antibody ratio (DAR) of 2, we explored both the reduction of the antibody with tris(2-carboxyethyl) phosphine (TCEP) as well as the use of a combination of glutathione and arginine as reducing and stabilizing agents, respectively. While exerting control over the DAR of the immunoconjugate proved cumbersome using TCEP, the use of glutathione and arginine enabled the selective reduction of the engineered cysteines and thus the formation of homogeneous immunoconjugates. A head-to-head comparison of the resulting ⁸⁹Zr-radioimmunoconjugates in mice bearing DLL3-expressing H82 xenografts revealed no significant differences in tumoral uptake and showed comparable radioactivity concentrations in most healthy nontarget organs. However, ⁸⁹Zr-DFO_PODS-^DAR2SC16-MB1 produced 30% lower uptake (3.3 ± 0.5 %ID/g) in the kidneys compared to ⁸⁹Zr-DFO_Mal-^DAR2SC16-MB1 (4.7 ± 0.5 %ID/g). In addition, H82-bearing mice injected with a ⁸⁹Zr-labeled isotype-control radioimmunoconjugate synthesized using PODS exhibited ∼40% lower radioactivity in the kidneys compared to mice administered its maleimide-based counterpart. Taken together, these results demonstrate the improved in vivo performance of the PODS-based radioimmunoconjugate and suggest that a stable, well-defined DAR2 radiopharmaceutical may be suitable for the clinical immunoPET of DLL3-expressing cancers.

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.

Substantial progress has been made in understanding how tumors escape immune surveillance. However, few measures to counteract tumor immune evasion have been developed. Suppression of tumor antigen expression is a common adaptive mechanism that cancers use to evade detection and destruction by the immune system. Epigenetic modifications play a critical role in various aspects of immune invasion, including the regulation of tumor antigen expression. To identify epigenetic regulators of tumor antigen expression, we established a transplantable syngeneic tumor model of immune escape with silenced antigen expression and used this system as a platform for a CRISPR-Cas9 suppressor screen for genes encoding epigenetic modifiers. We found that disruption of the genes encoding either of the chromatin modifiers activating transcription factor 7-interacting protein (Atf7ip) or its interacting partner SET domain bifurcated histone lysine methyltransferase 1 (Setdb1) in tumor cells restored tumor antigen expression. This resulted in augmented tumor immunogenicity concomitant with elevated endogenous retroviral (ERV) antigens and mRNA intron retention. ERV disinhibition was associated with a robust type I interferon response and increased T-cell infiltration, leading to rejection of cells lacking intact Atf7ip or Setdb1. ATF7IP or SETDB1 expression inversely correlated with antigen processing and presentation pathways, interferon signaling, and T-cell infiltration and cytotoxicity in human cancers. Our results provide a rationale for targeting Atf7ip or Setdb1 in cancer immunotherapy.