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Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.

Melanotransferrin (CD228/MFI2/MELTF) is a cell-surfaced glycosylphosphatidylinoitol (GPI)-anchored glycoprotein that belongs to the transferrin family of iron-binding proteins. CD228 was first described as an oncofetal protein highly expressed on malignant melanoma cells. Data from The Cancer Genome Atlas (TCGA) suggests that CD228 has broad expression across many types of carcinomas and it was recently described as a potential biomarker of invasive colorectal carcinoma. In this study, we characterize protein expression of CD228 using an immunohistochemical (IHC) assay and describe pre-clinical antitumor activity of SGN-CD228A, a potent CD228-targeting antibody-drug conjugate (ADC). We found that in addition to melanoma, CD228 is highly expressed in mesothelioma, non-small cell lung (NSCL), breast, colorectal, and pancreatic carcinomas. Monoclonal antibodies (mAbs) specific for human CD228 were evaluated and a lead antibody was selected based on binding characteristics, internalization properties, and cytotoxic activity as an ADC. SGN-CD228A is a humanized anti-CD228 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker, which incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous conjugation with decreased plasma clearance and increased preclinical antitumor activity. Interestingly, when evaluating drug linkers, we found that changing the linker from a di-peptide to β-glucuronidase resulted in a striking improvement in the cytotoxicity of MMAE, likely due to unique trafficking and recycling of CD228. We examined 50 carcinoma cell lines and found 41 had >10,000 CD228 receptors per cell of which 60% had EC50 values Citation Format: Sharsti L. Sandall, Marsha Mason, Devra Olson, Rebecca Mazahreh, Disha Sahetya, Lori Westendorf, Chris Leiske, Brian Schimpf, Liem Nguyen, Madhu Katepalli, Esther Trueblood, Christopher Hale, Albina Nesterova, Jason Wall, Timothy S. Lewis. SGN-CD228A: A novel humanized anti-CD228 antibody-drug conjugate for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2688.