Summary Investigating therapeutic “outliers” that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D “knock-in” mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency are more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer, and modulate competitive fitness and MEK dependency.

Infection with the human gastric pathogen Helicobacter pylori is associated with a spectrum of diseases including gastritis, peptic ulcers, gastric adenocarcinoma, and gastric mucosa–associated lymphoid tissue lymphoma. The cytotoxin-associated gene A (CagA) protein of H. pylori, which is translocated into host cells via a type IV secretion system, is a major risk factor for disease development. Experiments in gastric tissue culture cells have shown that once translocated, CagA activates the phosphatase SHP-2, which is a component of receptor tyrosine kinase (RTK) pathways whose over-activation is associated with cancer formation. Based on CagA's ability to activate SHP-2, it has been proposed that CagA functions as a prokaryotic mimic of the eukaryotic Grb2-associated binder (Gab) adaptor protein, which normally activates SHP-2. We have developed a transgenic Drosophila model to test this hypothesis by investigating whether CagA can function in a well-characterized Gab-dependent process: the specification of photoreceptors cells in the Drosophila eye. We demonstrate that CagA expression is sufficient to rescue photoreceptor development in the absence of the Drosophila Gab homologue, Daughter of Sevenless (DOS). Furthermore, CagA's ability to promote photoreceptor development requires the SHP-2 phosphatase Corkscrew (CSW). These results provide the first demonstration that CagA functions as a Gab protein within the tissue of an organism and provide insight into CagA's oncogenic potential. Since many translocated bacterial proteins target highly conserved eukaryotic cellular processes, such as the RTK signaling pathway, the transgenic Drosophila model should be of general use for testing the in vivo function of bacterial effector proteins and for identifying the host genes through which they function.

Alkaline phosphatases (Alps) are well-studied enzymes that remove phosphates from a variety of substrates. Alps function in diverse biological processes, including modulating host-bacterial interactions by dephosphorylating the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). In animals, Alps are encoded by multiple genes characterized by either ubiquitous expression (named Alpls for their liver expression, but a key to proper bone mineralization), or their tissue-specific expression, for example in the intestine (Alpi). We previously characterized a zebrafish alpi gene (renamed here alpi.1) that is regulated by Myd88-dependent innate immune signaling and that is required to prevent a host’s excessive inflammatory reactions to its resident microbiota. Here we report the characterization of two new alp genes in zebrafish, alpi.2 and alp3. To understand their origins, we investigated the phylogenetic history of Alp genes in animals. We find that vertebrate Alp genes are organized in three clades with one of these clades missing from the mammals. We present evidence that these three clades originated during the two vertebrate genome duplications. We show that alpl is ubiquitously expressed in zebrafish, as it is in mammals, whereas the other three alps are specific to the intestine. Our phylogenetic analysis reveals that in contrast to Alpl, which has been stably maintained as a single gene throughout the vertebrates, the Alpis have been lost and duplicated multiple times independently in vertebrate lineages, likely reflecting the rapid and dynamic evolution of vertebrate gut morphologies, driven by changes in bacterial associations and diet.

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Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed low glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited markedly reduced GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.