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. 2022 Oct 26;12(11):1570.
doi: 10.3390/biom12111570.

Replicative Instability Drives Cancer Progression

Benjamin B Morris  1   2 , Jason P Smith  1   3 , Qi Zhang  4 , Zhijie Jiang  4 , Oliver A Hampton  4 , Michelle L Churchman  4 , Susanne M Arnold  5 , Dwight H Owen  6 , Jhanelle E Gray  7 , Patrick M Dillon  8 , Hatem H Soliman  9 , Daniel G Stover  6 , Howard Colman  10 , Arnab Chakravarti  11 , Kenneth H Shain  12 , Ariosto S Silva  13 , John L Villano  5 , Michael A Vogelbaum  14 , Virginia F Borges  15 , Wallace L Akerley  16 , Ryan D Gentzler  8 , Richard D Hall  8 , Cindy B Matsen  17 , C M Ulrich  18 , Andrew R Post  19 , David A Nix  20 , Eric A Singer  21 , James M Larner  22 , Peter Todd Stukenberg  1 , David R Jones  23 , Marty W Mayo  1
Affiliations

Replicative Instability Drives Cancer Progression

Benjamin B Morris et al. Biomolecules. .

Abstract

In the past decade, defective DNA repair has been increasingly linked with cancer progression. Human tumors with markers of defective DNA repair and increased replication stress exhibit genomic instability and poor survival rates across tumor types. Seminal studies have demonstrated that genomic instability develops following inactivation of BRCA1, BRCA2, or BRCA-related genes. However, it is recognized that many tumors exhibit genomic instability but lack BRCA inactivation. We sought to identify a pan-cancer mechanism that underpins genomic instability and cancer progression in BRCA-wildtype tumors. Methods: Using multi-omics data from two independent consortia, we analyzed data from dozens of tumor types to identify patient cohorts characterized by poor outcomes, genomic instability, and wildtype BRCA genes. We developed several novel metrics to identify the genetic underpinnings of genomic instability in tumors with wildtype BRCA. Associated clinical data was mined to analyze patient responses to standard of care therapies and potential differences in metastatic dissemination. Results: Systematic analysis of the DNA repair landscape revealed that defective single-strand break repair, translesion synthesis, and non-homologous end-joining effectors drive genomic instability in tumors with wildtype BRCA and BRCA-related genes. Importantly, we find that loss of these effectors promotes replication stress, therapy resistance, and increased primary carcinoma to brain metastasis. Conclusions: Our results have defined a new pan-cancer class of tumors characterized by replicative instability (RIN). RIN is defined by the accumulation of intra-chromosomal, gene-level gain and loss events at replication stress sensitive (RSS) genome sites. We find that RIN accelerates cancer progression by driving copy number alterations and transcriptional program rewiring that promote tumor evolution. Clinically, we find that RIN drives therapy resistance and distant metastases across multiple tumor types.

Keywords: MYBL2; cancer progression; metastasis; replicative instability (RIN); single-strand break repair; translesion synthesis.

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Conflict of interest statement

J.P.S., Q.Z., Z.J., O.A.H., M.L.C., A.S., S.M.A., W.L.A., M.A.V., P.T.S., A.R.P., H.C., V.F.B., J.M.L., A.C., J.L.V., E.A.S., P.M.D., C.B.M., C.M.U., and D.A.N. have no conflict of interest relevant to this study to disclose. B.B.M. and M.W.M. hold provisional patent Serial No. 63/252,007. K.H.S. has consultancy, advisor, and/or speaker roles with Adaptive Biotech, Janssen, Bristol-Myers Squibb, Takeda, Sanofi, Glaxo Smith Kline, and Amgen. He has research funding with Karyopharm and Abbvie, and funds from BMS and Janssen for clinical trials. DHO is supported in part by research funding from BMS, Merck, Genetech, Pfizer, Onc.AI, and Palobiofarma provided to OSUCC. DGS serves on the Novartis advisory board. HHS has consultancy roles with Novartis, Astrazeneca, Eisai, PUMA, Seattle Genetics, and Sanofi. He also is supported in part by research funding to Moffitt Cancer Center provided by Amgen. D.R.J. has a consultancy and advisory roles at AstraZeneca as well as a clinical trial steering committee role at Merck. R.D.G. has consultancy, advisor, and/or speaker roles with Daiichi Sankyo, AstraZeneca, BluePrint Medicines, Pfizer, Mirati, Sanofi, Oncocyte, Jazz Pharmaceuticals, Rockepoint CME, Targeted Oncology, Total Health Conferencing, and OncLive. He is supported in part by research funding provided to UVA Comprehensive Cancer Center by Pfizer, Mirati, Daiichi Sankyo, Jounce Therapeutics, Helsinn, BMS, Merck, Janssen, and RTI International. D.G.S. has advisory roles for Novartis. J.E.G. has consultancy or advisory roles with AbbVie, AstraZeneca, Axiom HC Strategies, Blueprint Medicines, BMS, Celgene Copr, Diaachi Sankyo, EMD Serono, Genentech, Inivata, Janssen, Jazz Pharmaceuticals, Loxo Oncology, Merck, Norvartis, Sanofi, Takeda, OncoCyte, and Triptych Health Partners. She is supported in part by research funding provided by AstraZeneca, Boehringer Ingelheim, BMS, Genentech, G1 Therapeutics, Ludwig Institute of Cancer Research, Merck, Norvartis, and Pfizer. RGH has consultancy or advisory roles with BMS and Ono Pharmaceutical. He is supported in part by research funding to UVACC provided by Merck, AstraZeneca/MedImmune, Mirati Therapeutics, Lilly, and Daiichi Sankyo.

Figures

Figure 1
Figure 1
Elevated MYBL2 mRNA expression identifies patients with poor outcomes across multiple tumor types and genotypes. (A) Pan-cancer analysis overview. (B) Kaplan-Meier analyses demonstrate that MYBL2 expression is robustly prognostic across multiple tumor types for OS, DSS, and PFS outcomes. Log-rank test p-values are displayed. (C) MYBL2 High tumors develop across common cancer genetic driver backgrounds. Percentages reflect the percent of tumors with gene specific alterations. Statistical significance mapping represents Benjamini-Hochberg corrected q values, q < 0.05 *, q < 0.001 ***. (D) Individual tumors show different patterns of tumor suppressor inactivation and oncogene activation with respect to MYBL2 High and MYBL2 Low disease. IDHMUT LGG tumor suppressor and oncogene status were mapped excluding founding IDH mutations. One-sided Fisher’s exact test, p < 0.05 *, p < 0.01 **, ns: not significant. LUAD: lung adenocarcinoma. IDHMUT LGG: IDH-mutant lower grade glioma. PAAD: pancreatic adenocarcinoma. UCEC: uterine corpus endometrial carcinoma. SARC: sarcoma.
Figure 2
Figure 2
MYBL2 High tumors exhibit genomic instability despite containing wildtype BRCA genes. (A) MYBL2 High tumors have significantly greater somatic mutation burdens and fraction of the genome (FGA) altered. (B) MYBL2 High tumors have elevated microsatellite instability scores. (C) MYBL2 High tumors exhibit inefficient homologous recombination despite containing wildtype BRCA genes. (A–C) Statistical significance was assessed using Wilcoxon signed rank tests. p < 0.05 *, p < 0.01 **, p < 0.001 ***. (C) Enrichments for inactivating alterations in BRCA genes were tested using one-sided Fisher’s exact tests. Significance is mapped using Benjamini-Hochberg corrected q values. q < 0.05 *, q < 0.001 ***; ns, not significant.
Figure 3
Figure 3
Heterozygous losses impacting key DNA repair effectors are enriched in MYBL2 High tumors. (A) Weighted expression scores reveal an imbalance in DNA repair pathway regulation. (B) Observed differences in WE scores are highly correlated across different cancer types. Correlations with x marks indicate correlations that are not statistically significant (Pearson). (C) Heterozygous losses in genes encoding key single-strand break repair, TLS, and NHEJ effectors are highly enriched in MYBL2 High tumors. One-sided Fisher’s exact test, p < 0.05, *; p < 0.01, **; p < 0.001, ***. Heterozygous loss events are highly correlated with decreased expression of repair effectors. Benjamini-Hochberg corrected q. q < 0.05, *; q < 0.01, **, q < 0.001, ***. (D) COSMIC v3.2 SBS analysis reveals heterozygous loss of repair effectors is associated with impaired pathway function. S: Signatures specifically observed only in MYBL2 High or MYBL2 Low tumors. Dotted line represents Student’s t-test p = 0.05.
Figure 4
Figure 4
MYBL2 High tumors exhibit markers of chronic replication stress. (A) MYBL2 High tumors universally demonstrate significantly elevated replication stress scores. Wilcoxon, p < 0.001, ***. (B) MYBL2 High tumors experience a shift in intragenic somatic mutation position, relative to MYBL2 Low tumors. Kolmogorov-Smirnov test, p < 0.05, *; p < 0.01, **; p < 0.001, ***. (C) MYBL2 High tumors acquire significantly greater numbers of alterations at replication stress sensitive genomic sites. Wilcoxon, p < 0.01, **; p < 0.001, ***. ERFS sites [29], MiDAS sites [30].
Figure 5
Figure 5
Recurrent copy number alterations at replication stress sensitive sites rewire transcriptional programs and dysregulate master effectors controlling several hallmarks of cancer. (A) MYBL2 High tumors acquire copy number alterations in essential enzymes encoded at replication stress sensitive sites. (B) Enriched copy number alterations observed in MYBL2 High tumors rewire transcriptional programs and dysregulate master effectors controlling several hallmarks of cancer. Statistical significance is mapped according to Benjamini-Hochberg corrected q values. q < 0.05, *; q < 0.001, ***. Circled cluster numbers map to those displayed in (A). (C) Replication stress dysregulates master effectors controlling several hallmarks of cancer.
Figure 6
Figure 6
Elevated MYBL2 expression identifies patients at risk for poor responses to therapy and distant metastases across tumor types. (A) MYBL2 High patients have significantly poorer outcomes when treated with chemotherapy and irradiation regimens. Log-rank test p-values are displayed. (B) MYBL2 High tumors metastasize to distant sites at a higher frequency, including to the brain. (C) MYBL2 expression stratifies patient risk at diagnosis for brain metastasis development. LUAD: lung adenocarcinoma. ID-BRE: Invasive ductal breast cancer. IDHMUT LGG: IDH-mutant lower grade glioma. LRMM: Late relapse multiple myeloma. (D) MYBL2 expression is increased in brain metastases compared to patient matched primary lung adenocarcinoma tumors. (E) Replicative instability (RIN) accelerates genome evolution, driving cancer progression.
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