Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (As III ) is also an effective chemotherapeutic agent. The current use of As III in chemotherapy is limited to acute promyelocytic leukemia (APL). However, As III was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPRbased approach to identify modulators of As III toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased As III tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to As III whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to As III . Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to As III . Our data suggest a model in which an intracellular interaction between selenium and As III may impact intracellular As III levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of As III in CML cells. Targeting such processes simultaneously with As III treatment could potentiate As III in CML therapy.

Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.

Iron homeostasis is tightly regulated, and the peptide hormone hepcidin is considered to be a principal regulator of iron metabolism. Previous studies in a limited number of mouse strains found equivocal sex- and strain-dependent differences in mRNA and serum levels of hepcidin and reported conflicting data on the relationship between hepcidin () mRNA levels and iron status. Our aim was to clarify the relationships between strain, sex, and hepcidin expression by examining multiple tissues and the effects of different dietary conditions in multiple inbred strains. Two studies were done: first, mRNA, liver iron, and plasma diferric transferrin levels were measured in 14 inbred strains on a control diet; and second, mRNA and plasma hepcidin levels in both sexes and iron levels in the heart, kidneys, liver, pancreas, and spleen in males were measured in nine inbred/recombinant inbred strains raised on an iron-sufficient or high-iron diet. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). However, liver iron and diferric transferrin levels are not predictors of mRNA levels in mice fed iron-sufficient or high-iron diets, nor are the mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males. We also measured plasma erythroferrone, performed RNA-sequencing analysis of liver samples from six inbred strains fed the iron-sufficient, low-iron, or high-iron diets, and explored differences in gene expression between the strains with the highest and lowest hepcidin levels. Both sex and strain have a significant effect on both hepcidin mRNA (primarily a sex effect) and plasma hepcidin levels (primarily a strain effect). Liver iron and diferric transferrin levels are not predictors of mRNA levels in mice, nor are the mRNA and plasma hepcidin levels good predictors of tissue iron levels, at least in males.

Understanding how differentiation, microenvironment, and hormonal milieu influence human breast cell susceptibility to malignant transformation will require the use of physiologically relevant in vitro systems. We sought to develop a 3D culture model that enables the propagation of normal estrogen receptor alpha (ER)+ cells. We tested soluble factors and protocols for the ability to maintain progenitor and ER+ cells in cultures established from primary cells. Long-term representation of ER+ cells was optimal in medium that included three different TGFβ/activ β/activin activin receptor-like kinase inhibitors. We found that omitting the BMP signaling antagonist, Noggin, enhanced the responsiveness of the PGR gene to estradiol exposure without altering the proportions of ER+ cells in the cultures. Profiling of estradiol-exposed cultures showed that while all the cultures showed immediate and robust induction of PGR, LRP2, and IGFβ/activ B4, other responses varied qualitatively and quantitatively across specimens. We propose that these 3D cultures will overcome limitations of conventional 2D cultures of partially or fully transformed cell lines by sustaining normal endocrine function and growth regulation of the cell populations that comprise intact breasts.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.