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Supplementary Figures 1-8

Supplementary Methods Tables 1 and 2

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL.Significance:Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL.This article is highlighted in the In This Issue feature, p. 305

Understanding the intricacies of lethal prostate cancer poses specific challenges due to difficulties in accurate modeling of metastasis in vivo. Here we show that NPKEYFP mice (for Nkx3.1CreERT2/+; Ptenflox/flox; KrasLSL-G12D/+; R26R-CAG-LSL-EYFP/+) develop prostate cancer with a high penetrance of metastasis to bone, thereby enabling detection and tracking of bone metastasis in vivo and ex vivo. Transcriptomic and whole-exome analyses of bone metastasis from these mice revealed distinct molecular profiles conserved between human and mouse and specific patterns of subclonal branching from the primary tumor. Integrating bulk and single-cell transcriptomic data from mouse and human datasets with functional studies in vivo unravels a unique MYC/RAS co-activation signature associated with prostate cancer metastasis. Finally, we identify a gene signature with prognostic value for time to metastasis and predictive of treatment response in human patients undergoing androgen receptor therapy across clinical cohorts, thus uncovering conserved mechanisms of metastasis with potential translational significance. Using lineage tracing and molecular profiling, Abate-Shen and colleagues identify a Ras and Myc co-activation signature that predicts metastasis and castration resistance in localized prostate cancer.

Brain metastasis is a major cause of morbidity and mortality in cancer patients. Here we investigated mechanisms allowing small-cell lung cancer (SCLC) cells to grow in the brain. We show that SCLC cells undergo a cell state transition towards neuronal differentiation during tumor progression and metastasis, and that this neuronal mimicry is critical for SCLC growth in the brain. Mechanistically, SCLC cells re-activate astrocytes, which in turn promote SCLC growth by secreting neuronal pro-survival factors such as SERPINE1. We further identify Reelin, a molecule important in brain development, as a factor secreted by SCLC cells to recruit astrocytes to brain metastases in mice. This recruitment of astrocytes by SCLC was recapitulated in assembloids between SCLC aggregates and human cortical spheroids. Thus, SCLC brain metastases grow by co-opting mechanisms involved in reciprocal neuron-astrocyte interactions during development. Targeting such developmental programs activated in this cancer ecosystem may help treat brain metastases.

Summary: Small cell lung cancer (SCLC) is an aggressive disease with dismal survival rates and limited therapeutic options. SCLC development is strongly associated with exposure to tobacco carcinogens. However, additional genetic and environmental risk factors that contribute to SCLC pathogenesis are beginning to emerge. Here, we specifically assess disparities pertaining to SCLC in Black populations. In contrast to non–small cell lung cancer, preliminary data suggest that Black individuals may actually be at a lower risk of developing SCLC relative to white individuals. This difference remains unexplained but urgently needs to be verified in larger data sets, because it could provide important new insights and approaches to understanding this recalcitrant tumor. Importantly, little biological information exists on SCLC in Black individuals, and few patient-derived preclinical SCLC models from diverse ancestries are available in the laboratory. Unfortunately, we note strikingly low numbers of Black participants in clinical trials testing new treatments for SCLC. Evidence further indicates that care for patients with SCLC may vary between communities with a large fraction of Black patients and those without. Together, these observations underscore the need to better investigate genetic, environmental, and socioeconomic factors associated with SCLC development, preclinical research, clinical care, and outcomes.

Relapsed acute lymphoblastic leukaemia (ALL) is associated with resistance to chemotherapy and poor prognosis. Gain-of-function mutations in the 5'-nucleotidase, cytosolic II (NT5C2) gene induce resistance to 6-mercaptopurine and are selectively present in relapsed ALL. Yet, the mechanisms involved in NT5C2 mutation-driven clonal evolution during the initiation of leukaemia, disease progression and relapse remain unknown. Here we use a conditional-and-inducible leukaemia model to demonstrate that expression of NT5C2(R367Q), a highly prevalent relapsed-ALL NT5C2 mutation, induces resistance to chemotherapy with 6-mercaptopurine at the cost of impaired leukaemia cell growth and leukaemia-initiating cell activity. The loss-of-fitness phenotype of NT5C2+/R367Q mutant cells is associated with excess export of purines to the extracellular space and depletion of the intracellular purine-nucleotide pool. Consequently, blocking guanosine synthesis by inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) induced increased cytotoxicity against NT5C2-mutant leukaemia lymphoblasts. These results identify the fitness cost of NT5C2 mutation and resistance to chemotherapy as key evolutionary drivers that shape clonal evolution in relapsed ALL and support a role for IMPDH inhibition in the treatment of ALL.

The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL. Significance: Phf6 controls HSC homeostasis, leukemia initiation, and T-ALL leukemia-initiating cell self-renewal. These results substantiate a role for PHF6 mutations as early events and drivers of leukemia stem cell activity in the pathogenesis of T-ALL. This article is highlighted in the In This Issue feature, p. 305

Cancer is caused by germline and somatic mutations, which can share biological features such as amino acid change. However, integrated germline and somatic analysis remains uncommon. We present a framework that uses machine learning to learn features of recurrent somatic mutations to (1) predict somatic variants from tumor-only samples and (2) identify somatic-like germline variants for integrated analysis of tumor-normal DNA. Using data from 1769 patients from seven cancer types (bladder, glioblastoma, low-grade glioma, lung, melanoma, stomach, and pediatric glioma), we show that “somatic-like” germline variants are enriched for autosomal-dominant cancer-predisposition genes (p, Cancer: Analytic tool reveals inherited and non-inherited tumor-causing alterations Bladder cancer cells often harbor DNA mutations that occur after tumor development, including some mutations that affect DNA repair. Raul Rabadan, Jiguang Wang, and colleagues from Columbia University in New York, USA, developed an analytic framework for identifying genetic variants, both inherited and newly arisen, that contribute to tumor development. The machine-learning tool—known as Tumor-Only Boosting Identification, or TOBI—learns what’s a cancer-associated mutation from a small training set of tumor samples and matched healthy controls. The researchers then fed the algorithm data from the tumors of 1769 patients with cancers of the bladder, brain, lungs, stomach and skin. They found that TOBI pinpointed many inherited and non-inherited mutations known to contribute to cancer growth. In bladder cancer samples, the tool also revealed a previously unknown role for inherited mutations in BRCA2 and other DNA repair genes in the so-called Fanconi anemia pathway.

To ensure the completion of DNA replication and maintenance of genome integrity, DNA repair factors protect stalled replication forks upon replication stress. Previous studies have identified a critical role for the tumor suppressors BRCA1 and BRCA2 in preventing the degradation of nascent DNA by the MRE11 nuclease after replication stress. Here we show that depletion of SMARCAL1, a SNF2-family DNA translocase that remodels stalled forks, restores replication fork stability and reduces the formation of replication stress-induced DNA breaks and chromosomal aberrations in BRCA1/2-deficient cells. In addition to SMARCAL1, other SNF2-family fork remodelers, including ZRANB3 and HLTF, cause nascent DNA degradation and genomic instability in BRCA1/2-deficient cells upon replication stress. Our observations indicate that nascent DNA degradation in BRCA1/2-deficient cells occurs as a consequence of MRE11-dependent nucleolytic processing of reversed forks generated by fork remodelers. These studies provide mechanistic insights into the processes that cause genome instability in BRCA1/2- deficient cells.

Despite large-scale cancer genomics studies, key somatic mutations driving cancer, and their functional roles, remain elusive. Here we propose that analysis of comorbidities of Mendelian diseases with cancers provides a novel, systematic way to discover new cancer genes. If germline genetic variation in Mendelian loci predisposes bearers to common cancers, the same loci may harbor cancer-associated somatic variation. Compilations of clinical records spanning over 100 million patients provide an unprecedented opportunity to assess clinical associations between Mendelian diseases and cancers. We systematically compare these comorbidities against recurrent somatic mutations from more than five thousand patients across many cancers. Using multiple measures of genetic similarity, we show that a Mendelian disease and comorbid cancer indeed have genetic alterations of significant functional similarity. This result provides a basis to identify candidate drivers in cancers including melanoma and glioblastoma. Some Mendelian diseases demonstrate “pan-cancer” comorbidity and shared genetics across cancers.

The evolutionary history and epidemiology of parasites may be reflected in the extent and geographic distribution of their genetic variation. Among coccidian parasites, the population structure of only Toxoplasma gondii has been extensively examined. Intraspecific variation in other coccidia, for example, those assigned to the genus Besnoitia, remains poorly defined. Here, we characterize the extent of genetic variation among populations of Besnoitia tarandi , a parasite whose intermediate hosts include reindeer/caribou ( Rangifer tarandus ). Isolates from the Canadian Arctic and Finnish sub-Arctic were genotyped at six microsatellite loci, the first internal transcribed spacer region of nuclear rDNA, and the RNA polymerase β subunit ( rpoB ) encoded in the plastid genome. Remarkably, all isolates exhibited the same multilocus genotype, regardless of the isolate’s geographic origin. This absolute monomorphism occurred despite the capacity of these loci to vary, as established by evident differentiation between B. tarandi and two other species of Besnoitia, and variation among four isolates of B. besnoiti . The surprising lack of genetic variation across the sampled range suggests that B. tarandi may have experienced a recent population bottleneck.