Cancer evolves through the emergence and selection of molecular alterations. Cancer genome profiling has revealed that specific events are more or less likely to be co-selected, suggesting that the selection of one event depends on the others. However, the nature of these evolutionary dependencies and their impact remain unclear. Here, we designed SELECT, an algorithmic approach to systematically identify evolutionary dependencies from alteration patterns. By analyzing 6,456 genomes from multiple tumor types, we constructed a map of oncogenic dependencies associated with cellular pathways, transcriptional readouts, and therapeutic response. Finally, modeling of cancer evolution shows that alteration dependencies emerge only under conditional selection. These results provide a framework for the design of strategies to predict cancer progression and therapeutic response.

Hi-C chromatin maps of EZH2 WT and EZH2 Y646X lymphomas. EZH2 p.Tyr646* gain-of-function alterations lead to a genomewide increase in H3K27me3 (Supplementary Fig. 1a). To establish whether this global accumulation of H3K27me3 modifies the genome topology on a similarly broad scale, we performed high-throughput chromatin conformation capture (Hi-C) in two lymphoma cell lines (Karpas422 and WSU-DLCL2) expressing the mutant form of EZH2 (EZH2 Y646X) and a lymphoma cell line (OCI-Ly19) expressing the wild-type EZH2 (EZH2 WT) protein (Supplementary Table 1 and Supplementary Note). Contact maps of EZH2 Y646X and EZH2 WT cells were binned in regions of 50 kb and compared with multiple metrics (Fig. 1). For each pair of maps, we compared the overall distribution of chromosomal contacts by using the stratum-adjusted correlation coefficient (SCC) 24 (Fig. 1a); the fraction of 1-Mb regions assigned to the same compartment (A or B) 3 (Fig. 1b); the similarity among TADs 25-27 (Fig. 1c); and the fraction of bin interactions that were significant in both maps, also known as the cell interactome 28,29 (Fig. 1d). To build a reference scale of values for each metric, we compared Hi-C maps of EZH2 Y646X lymphoma cell lines with Hi-C maps of endothelial cells (HUVEC), fetal fibroblasts (IMR90), and normal lymphoblastoid cells (GM12878). Moreover, we used randomized contact maps or

Cancer evolution determines molecular and morphological intra-tumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histological features remains elusive. Here, we generated multi-omics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intra-tumor spatial heterogeneity, tracing non-genetic routes of cancer evolution. Statement of significance Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized non-genetic mechanisms underlying intra-tumor patterns and molecular markers predicting patient prognosis. Intra-tumor patterns determined diverse immune microenvironments warranting their study in the context of current immunotherapies.

Highlights d CTSS is overexpressed and mutated (Y132D) in follicular lymphoma d CTSS regulates antigen processing and communication with CD4 + Tfh cells d Loss of CTSS activity promotes CD8 + T cell infiltration d Alteration of CTSS-mediated antigen processing contributes to antigen diversification

Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2. Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.

In diffuse large B-cell lymphoma (DLBCL), activation of the B-cell receptor (BCR) promotes multiple oncogenic signals, which are essential for tumor proliferation. Inhibition of the Bruton's tyrosine kinase (BTK), a BCR downstream target, is therapeutically effective only in a subgroup of patients with DLBCL. Here, we used lymphoma cells isolated from patients with DLBCL to measure the effects of targeted therapies on BCR signaling and to anticipate response. In lymphomas resistant to BTK inhibition, we show that blocking BTK activity enhanced tumor dependencies from alternative oncogenic signals downstream of the BCR, converging on MYC upregulation. To completely ablate the activity of the BCR, we genetically and pharmacologically repressed the activity of the SRC kinases LYN, FYN, and BLK, which are responsible for the propagation of the BCR signal. Inhibition of these kinases strongly reduced tumor growth in xenografts and cell lines derived from patients with DLBCL independent of their molecular subtype, advancing the possibility to be relevant therapeutic targets in broad and diverse groups of DLBCL patients.

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<div>Abstract<p>Cancer evolution determines molecular and morphologic intratumor heterogeneity and challenges the design of effective treatments. In lung adenocarcinoma, disease progression and prognosis are associated with the appearance of morphologically diverse tumor regions, termed histologic patterns. However, the link between molecular and histologic features remains elusive. Here, we generated multiomics and spatially resolved molecular profiles of histologic patterns from primary lung adenocarcinoma, which we integrated with molecular data from >2,000 patients. The transition from indolent to aggressive patterns was not driven by genetic alterations but by epigenetic and transcriptional reprogramming reshaping cancer cell identity. A signature quantifying this transition was an independent predictor of patient prognosis in multiple human cohorts. Within individual tumors, highly multiplexed protein spatial profiling revealed coexistence of immune desert, inflamed, and excluded regions, which matched histologic pattern composition. Our results provide a detailed molecular map of lung adenocarcinoma intratumor spatial heterogeneity, tracing nongenetic routes of cancer evolution.</p>Significance:<p>Lung adenocarcinomas are classified based on histologic pattern prevalence. However, individual tumors exhibit multiple patterns with unknown molecular features. We characterized nongenetic mechanisms underlying intratumor patterns and molecular markers predicting patient prognosis. Intratumor patterns determined diverse immune microenvironments, warranting their study in the context of current immunotherapies.</p><p><i>This article is highlighted in the In This Issue feature, p. 1307</i></p></div>