ABT-199, a potent and selective small-molecule antagonist of BCL-2, is being clinically vetted as pharmacotherapy for the treatment of acute myeloid leukemia (AML). However, given that prolonged monotherapy tends to beget resistance, we sought to investigate the means by which resistance to ABT-199 might arise in AML and the extent to which those mechanisms might be preempted. Here we used a pathway-activating genetic screen to nominate MCL-1 and BCL-XL as potential nodes of resistance. We then characterized a panel of ABT-199-resistant myeloid leukemia cell lines derived through chronic exposure to ABT-199 and found that acquired drug resistance is indeed driven by the upregulation of MCL-1 and BCL-XL. By targeting MCL-1 and BCL-XL, resistant AML cell lines could be resensitized to ABT-199. Further, preemptively targeting MCL-1 and/or BCL-XL alongside administration of ABT-199 was capable of delaying or forestalling the acquisition of drug resistance. Collectively, these data suggest that in AML, (1) the selection of initial therapy dynamically templates the landscape of acquired resistance via modulation of MCL-1/BCL-XL and (2) appropriate selection of initial therapy may delay or altogether forestall the acquisition of resistance to ABT-199.

Multiple species within the basidiomycete genus Cryptococcus cause cryptococcal disease. These species are estimated to affect nearly a quarter of a million people leading to ∼180,000 mortalities, annually. Sexual reproduction, which can occur between haploid yeasts of the same or opposite mating type, is a potentially important contributor to pathogenesis as recombination can generate novel genotypes and transgressive phenotypes. However, our quantitative understanding of recombination in this clinically important yeast is limited. Here, we describe genome-wide estimates of recombination rates in Cryptococcus deneoformans and compare recombination between progeny from α–α unisexual and a–α bisexual crosses. We find that offspring from bisexual crosses have modestly higher average rates of recombination than those derived from unisexual crosses. Recombination hot and cold spots across the C. deneoformans genome are also identified and are associated with increased GC content. Finally, we observed regions genome-wide with allele frequencies deviating from the expected parental ratio. These findings and observations advance our quantitative understanding of the genetic events that occur during sexual reproduction in C. deneoformans, and the impact that different forms of sexual reproduction are likely to have on genetic diversity in this important fungal pathogen.

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a “function-valued” QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation.

Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance tness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be bene cial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~ 15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identi ed. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the rst exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identi ed a signi cant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identified. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the first exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identified a significant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

Cellular development is orchestrated by evolutionarily conserved signaling pathways, which are often pleiotropic and involve intra- and inter-pathway epistatic interactions that form intricate, complex regulatory networks. Cryptococcus species are a group of closely-related human fungal pathogens that grow as yeasts yet transition to hyphae during sexual reproduction. Additionally, during infection they can form large, polyploid titan cells that evade immunity and develop drug resistance. Multiple known signaling pathways regulate cellular development, yet how these are coordinated and interact with genetic variation is less well understood. Here, we conducted quantitative trait locus (QTL) analyses of a mapping population generated by sexual reproduction of two parents, only one of which is unisexually fertile. We observed transgressive segregation of the unisexual phenotype among progeny, as well as a novel large-cell phenotype under mating-inducing conditions. These large-cell progeny were found to produce titan cells both in vitro and in infected animals. Two major QTLs and corresponding quantitative trait genes (QTGs) were identified: RIC8 (encoding a guanine-exchange factor) and CNC06490 (encoding a putative Rho-GTPase activator), both involved in G-protein signaling. The two QTGs interact epistatically with each other and with the mating-type locus in phenotypic determination. These findings provide insights into the complex genetics of morphogenesis during unisexual reproduction and pathogenic titan cell formation and illustrate how QTL analysis can be applied to identify epistasis between genes. This study shows that phenotypic outcomes are influenced by the genetic background upon which mutations arise, implicating dynamic, complex genotype-to-phenotype landscapes in fungal pathogens and beyond.

Multiple species within the basidiomycete genus, Cryptococcus, cause cryptococcal disease. These species are estimated to affect nearly a quarter of a million people leading to approximately 180,000 mortalities, annually. Sexual reproduction, which can occur between haploid yeasts of the same or opposite mating type, is a potentially important contributor to pathogenesis as recombination can generate novel genotypes and transgressive phenotypes. However, our quantitative understanding of recombination in this clinically important yeast is limited. Here we describe genome-wide estimates of recombination rates in Cryptococcus deneoformans and compare recombination between progeny from α-α unisexual and a-α bisexual crosses. We find that offspring from bisexual crosses have modestly higher average rates of recombination than those derived from unisexual crosses. Recombination hot and cold spots across the C. deneoformans genome are also identified and are associated with increased GC content. Finally, we observed regions genome-wide with allele frequencies deviating from the expected parental ratio. These findings and observations advance our quantitative understanding of the genetic events that occur during sexual reproduction in C. deneoformans, and the impact that different forms of sexual reproduction are likely to have on genetic diversity in this important fungal pathogen. (Kwon-Chung 1975, 1976 Litv-21 intseva et al. 2003; Lin et al. 2007;Hull et al. 2002) 107 Materials and Methods 108Strains, laboratory crosses and isolation 109As previously described (Sun et al. 2012(Sun et al. , 2014 . CC-BY-NC-ND4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/234617 doi: bioRxiv preprint first posted online Dec. 14, 2017; α-α unisexual crosses and 39 from a-α bisexual crosses, were re-167 tained for analysis.

Significance Cellular development is regulated by a complex web of signaling pathways that respond to both intracellular and extracellular cues. Morphological transitions in pathogenic fungi, such as those observed during sexual reproduction or in response to the host environment, offer tractable models for understanding the principles that govern eukaryotic cell development and morphogenesis. Using the human fungal pathogen Cryptococcus deneoformans as a model and applying quantitative trait locus analysis, we defined genes and gene–gene interactions involved in the yeast–hyphal transition and titanization, two morphological developments that are important for adaptation, pathogenesis, and evolution of this fungal pathogen. Our study highlights the conservation and complexity of key signaling pathways in regulating cell development in fungi, as well as other eukaryotes.