Elizabeth K. Benitez

Abstract As cancers progress, they become increasingly aggressive—metastatic tumours are less responsive to first-line therapies than primary tumours, they acquire resistance to successive therapies and eventually cause death 1,2 . Mutations are largely conserved between primary and metastatic tumours from the same patients, suggesting that non-genetic phenotypic plasticity has a major role in cancer progression and therapy resistance 3–5 . However, we lack an understanding of metastatic cell states and the mechanisms by which they transition. Here, in a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that, although primary tumours largely adopt LGR5 + intestinal stem-like states, metastases display progressive plasticity. Cancer cells lose intestinal cell identities and reprogram into a highly conserved fetal progenitor state before undergoing non-canonical differentiation into divergent squamous and neuroendocrine-like states, a process that is exacerbated in metastasis and by chemotherapy and is associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues compared with their intestinal lineage-restricted primary tumour counterparts. We identify PROX1 as a repressor of non-intestinal lineage in the fetal progenitor state, and show that downregulation of PROX1 licenses non-canonical reprogramming.

Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.

Abstract Background: The incidence of early onset (EO) colorectal cancer (CRC) (age <50) is rising with a left-sided predominance. Response to chemotherapy and anti-EGFR therapies differ in left- and right-sided colon cancers after accounting for genomic differences. The intestinal microbiome may contribute to CRC pathogenesis and response to therapy; how organisms drive metastasis and treatment resistance is not known. Purpose: To define the microbiome contribution to EO-CRC and response to treatment by analyzing longitudinal samples from previously untreated patients with CRC. Methods: We designed a prospective biospecimen collection platform and selected patients with previously untreated CRC. We collected stool, biopsy or surgical tissue, and peripheral blood mononuclear cells at baseline and serially throughout treatment. Stool samples were analyzed using shotgun sequencing. Alpha diversity was calculated using the inverse Simpson index and compared between groups using the Wilcoxon signed-rank test. Beta diversity was analyzed using the Bray-Curtis dissimilarity matrix and compared using PERMANOVA. Multivariate associations between species abundance, metabolic pathway abundance and clinical covariates were performed using MaAsLin2 R package. Results: We analyzed a total of 132 stool samples from 65 patients with CRC including up to 5 samples from a single patient over time. Mean alpha diversity did not differ significantly by primary site, stage or age at diagnosis. Beta diversity was significantly different between samples from right- compared with left-sided CRC (P=.011) and pre- and post-surgery (P=.001). Beta diversity also differed significantly by diagnosis age (<50 vs. > 50) (P=.001 all samples, P=.032 baseline samples). When CRC baseline samples were examined at the species level, Blautia glucerasea was associated with right-sided primary (P<.001, Q=.1). Considering non-metastatic cases, recurrence was associated with increased Enteroscipio rubneri, Actinomyces oral taxon 448, Bacteroidales sp. and Lancefieldella parvula (P<.01, Q>0.1). We further investigated metabolic pathway associations. The superpathway of L-threonine metabolism was significantly increased in samples from patients with right-sided colon cancer at baseline (P<.001, Q=.03). Considering samples across all time points, right-sided colon cancer was associated with increased abundance of pathways associated with propanoate degradation, L-threonine metabolism, yeast NADPH production, and hydroxyphenylacetate degradation (all P<.001, Q<.05). Conclusions: The functional effects of the intestinal microbiome may underlie the distinct biology of right-sided colon cancer. Composition of the intestinal microbiome differs by age at diagnosis. Updated recurrence data will be presented. Citation Format: Melissa Lumish, Nicholas Waters, Joshua Leinwand, Asha Saxena, Anqi Dai, Teng Fei, Maggie Fox, Andre Chavez, Jonathan Bermeo, Dorina Ismailgeci, Elizabeth Benitez, Christopher Cowley, Julio Garcia-Aguilar, Martin Weiser, Andrea Cercek, Luis A. Diaz, Marcel van den Brink, Jonathan Peled, Karuna Ganesh. Shotgun sequencing of serial fecal microbiome samples in patients with colorectal cancer reveals distinct bacterial species and metabolic pathways associated with tumor sidedness and age at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2805.