Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer.

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. "Drug-tolerant persisters" (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a "pre-DTP" state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor-dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation. SIGNIFICANCE: DTPs are implicated in resistance to anticancer therapies, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells ("pre-DTPs") that engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and potential therapeutic vulnerabilities. This article is highlighted in the In This Issue feature, p. 873.

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin ß6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin ß6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

Post-translational regulation of CD133 by ATase1/ATase2-mediated lysine acetylation.

The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.

Regulation of CD133 by HDAC6 promotes ß-catenin signaling to suppress cancer cell differentiation.

The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, ß-catenin, can physically associate as a ternary complex. This association stabilizes ß-catenin via HDAC6 deacetylase activity, which leads to activation of ß-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased ß-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

Small molecule antagonists of the Wnt/ß-catenin signaling pathway target breast tumor-initiating cells in a Her2/Neu mouse model of breast cancer.

BACKGROUND: Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure. METHODS/FINDINGS: To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/ß-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118-310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118-310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118-310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118-310 treated mice were unable to seed the growth of secondary tumors after transplant. CONCLUSIONS: These studies demonstrate that inhibitors of Wnt/ß-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.

The mixed lineage leukemia (MLL) fusion-associated gene AF4 promotes CD133 transcription.

The AC133 epitope has been used as a marker for both normal and cancer stem cells from multiple tissue lineages. To identify transcription factors that regulate CD133 expression, we conducted parallel large-scale RNA interference screens in Caco-2 cancer cells that endogenously express CD133 and in engineered HEK293 cells that express CD133 from a heterologous promoter. The transcription factor AF4 was identified following a comparative analysis between the two screens. We then showed that AF4 is a promoter of CD133 transcription in multiple cancer cell lines. Knockdown of AF4 resulted in a dramatic reduction in CD133 transcript levels. Importantly, a subset of pediatric acute lymphoblastic leukemias (ALL) harbor a fusion oncogene results from a chromosomal translocation that juxtaposes the mixed-lineage leukemia (MLL) gene and the AF4 gene. An investigation of the functional role of CD133 in the MLL-AF4-dependent ALL cells revealed that CD133 was required for leukemia cell survival. Together, our findings show AF4-dependent regulation of CD133 expression, which is required for the growth of ALL cells. CD133 may therefore represent a therapeutic target in a subset of cancers.