Chen, Claire1; Shih, Juliann1; Sutton, Jenna1; Cabugao, Paul2; Romero, Arthur1 Author Information

Figure S1. Super-enhancers near the KLF5 gene are focally amplified in cancer cell lines;Figure S2. The regulatory potential of the KLF5/KLF12 intervening region;Figure S3. Expression level of KLF12, DIS3 and PIBF1 after repression of KLF5 superenhancers;Figure S4. Genomic analysis of KLF5 binding sites in BICR31 cells;Figure S5. Target genes of KLF5 in head and neck squamous cell carcinomas;Figure S6. The effect of KLF5 silencing on H3K27ac profile;Figure S7. Mutation hotspots in KLF5;Figure S8. Mutation profile in FBXW7 in colorectal cancers; Figure S9. Ectopic expression of KLF5 mutants in HEK293T cells; Figure S10. Ectopic expression of KLF5 E419Q in HCC95 cells

Table S4 includes immune signature and iCluster results.

Chemical structures of compounds used in study

Table S7 contains microbe screening results.

Table S1 contains cohort description, Master Patient Table and MutSigCV results.

Supplementary Methods. Supplementary Figure 1. Somatic alterations in PPARG and RXRA are hallmarks of luminal bladder cancer. Supplementary Figure 2. PPARG pathway is activated by overexpression of RXRA S427F/S427Y, but not other mutant alleles in bladder cancer cells. Supplementary Figure 3. Representation of the effects of ligand-dependent modulation on the PPARG interactome. Supplementary Figure 4. Downregulation of FABP4 protein by treatment of PPARG-activated bladder cancer cell lines by inverse-agonist T0070907. Supplementary Figure 5. Genome engineering scheme for generating reporter cell line. Supplementary Figure 6. Basal expression of FABP4 is reduced by PPARG inverse agonists, but not antagonists. Supplementary Figure 7. Lipid metabolism genes are inhibited by PPARG inverse-agonists. Supplementary Figure 8. PPARG inverse-agonists inhibit proliferation of PPARG activated bladder cancer cell lines in clonogenic assays. Supplementary Figure 9. PPARG inverse-agonists, but not antagonists, inhibit proliferation of PPARG-activated bladder cancer cell lines. Supplementary Figure 10. Somatic alterations in RXRA and PPARG.

Table S2 contains BAP1 analysis results, as well as detailed lists of YY1 and IRF8 target genes.

Table S6 contains results from the analysis of DNA methylation in SETD2 mutated and BAP1 inactivated samples.

Supplementary materials and methods, as well as supplementary figures S1-S7.

Table S1: KLF5 E419Q-gained super-enhancers and the nearest genes

Aneuploidies—whole-chromosome or whole-arm imbalances—are the most prevalent alteration in cancer genomes 1 , 2 . However, it is still debated whether their prevalence is due to selection or ease of generation as passenger events 1 , 2 . Here we developed a method, BISCUT, that identifies loci subject to fitness advantages or disadvantages by interrogating length distributions of telomere- or centromere-bounded copy-number events. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage specific. BISCUT identified the helicase-encoding gene WRN as a haploinsufficient tumour-suppressor gene on chromosome 8p, which is supported by several lines of evidence. We also formally quantified the role of selection and mechanical biases in driving aneuploidy, finding that rates of arm-level copy-number alterations are most highly correlated with their effects on cellular fitness 1 , 2 . These results provide insight into the driving forces behind aneuploidy and its contribution to tumorigenesis.

Table S3 contains the karyotypes of 16 genome-wide LOH MPM cases from the BWH cohort.

Table S5 contains detailed miRs and lncRNAs results.

INTRODUCTION: Congenital bullous emphysema (CBE) is a rare disorder that affects an estimated 1/25,000 births, and is most commonly found in male children with bronchial and cardiovascular malformations.We present a case of CBE with atypical phenotype and multiple complications eventually requiring lobectomy. CASE PRESENTATION:A 52 year old female with a two pack year smoking history presented with cough and dyspnea.Respiratory biofire showed H. influenza infection, while imaging revealed large, bibasilar bullae with near complete effacement of the regional lung parenchyma and mosaic attenuation.The patient improved with standard treatment for an acute exacerbation of chronic obstructive pulmonary disease and only required 3L of supplemental oxygen on discharge; however, the size of the bullae and extent of the emphysema was inconsistent with her smoking history.Notably, the patient had a negative serum alpha antitrypsin, alpha-1 antitrypsin S allele, Z allele, SERPINA1 gene sequencing, antinuclear nuclear antitrypsin, antineutrophilic cytoplasmic antibody, autoimmune superpanel, histoplasma and aspergillus antigen.The patient denied any complications at birth, occupational exposure, or records of previous imaging.Unfortunately, the patient would present again multiple times for hydro-and pneumothoracies over the next two years.Ultimately, the patient underwent bullectomy with resection of the right middle and lower lobe.The procedure was complicated by right upper lobe collapse and tension pneumothorax, requiring bilateral chest tube placement.Despite heavy pulmonary trauma, the patient was discharged on only Stiolto (LAMA/LABA) and 3L of supplemental oxygen.Pathology only showed normal lung tissue with chronic inflammatory changes.The patient has not suffered another pneumothorax to this date.DISCUSSION: It is hypothesized that hypoplastic cartilaginous folds develop abnormal lung architecture that predispose children to recurrent pulmonary infections, eventually growing into hyperinflated bullae that compress surrounding lobes, compromise vascular flow, and can even cause mediastinal shift to the point of herniation into the contralateral hemithorax.Bullae are typically asymmetric, apical, paraseptal, and progressive, although our female patient had bibasilar, paraseptal bullae.CONCLUSIONS: CBE is a rare disorder that can be complicated with recurrent pneumothoracies.Lobectomy may prove preventative against pneumothorax, and bridge the patient to lung transplant evaluation.

Table S2: Oligos used in the study

The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

Aneuploidy, which we define as whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, head and neck, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although these alterations are frequent, they are not well understood due to difficulty in modeling specific aneuploidy alterations in the matching cell type. However, recent advances in genome engineering allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells most similar to upper airway basal cells. Deletion of chromosome 3p was validated by whole genome sequencing and karyotyping. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells. Genome sequencing and karyotype analyses found that evidence of chromosome 3 duplication, transitioning the cell to a state of chromosome 3q gain. We isolated sibling cells with chromosome 3p deletion or chromosome 3q gain and demonstrated that duplication of chromosome 3 could indeed rescue proliferation rates. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro, and a possible explanation for joint alteration of both arms of chromosome 3. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2135.

Aneuploidies, defined as whole-arm or whole-chromosome imbalances, are the most prevalent alteration in cancer genomes. However, the extent to which they are enriched due to selection is unclear, against the alternative hypothesis that they are passenger events that are simply highly prone to occur. We developed a novel method, BrISCUT, that identifies loci under selective advantage or disadvantage due to arm-level copy-number alterations by interrogating length distributions of events that are bounded at either the telomere or centromere. These loci were significantly enriched for known cancer driver genes, including genes not detected through analysis of focal copy-number events, and were often lineage-specific. We also formally quantified the role of selection and mechanistic biases in driving aneuploidy, finding that rates of arm-level SCNAs are most highly correlated with selective pressures. These results provide insight into the causes of aneuploidies and their contributions to tumorigenesis.