Homologous recombination deficiency signatures in gastrointestinal and thoracic cancers correlate with platinum therapy duration.

There is emerging evidence about the predictive role of homologous recombination deficiency (HRD), but this is less defined in gastrointestinal (GI) and thoracic malignancies. We reviewed whole genome (WGS) and transcriptomic (RNA-Seq) data from advanced GI and thoracic cancers in the Personalized OncoGenomics trial (NCT02155621) to evaluate HRD scores and single base substitution (SBS)3, which is associated with BRCA1/2 mutations and potentially predictive of defective HRD. HRD scores were calculated by sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores. Regression analyses examined the association between HRD and time to progression on platinum (TTPp). We included 223 patients with GI (n = 154) or thoracic (n = 69) malignancies. TTPp was associated with SBS3 (p < 0.01) but not HRD score in patients with GI malignancies, whereas neither was associated with TTPp in thoracic malignancies. Tumors with gBRCA1/2 mutations and a somatic second alteration exhibited high SBS3 and HRD scores, but these signatures were also present in several tumors with germline but no somatic second alterations, suggesting silencing of the wild-type allele or BRCA1/2 haploinsufficiency. Biallelic inactivation of an HR gene, including loss of XRCC2 and BARD1, was identified in BRCA1/2 wild-type HRD tumors and these patients had prolonged response to platinum. Thoracic cases with high HRD score were associated with high RECQL5 expression (p ≤ 0.025), indicating another potential mechanism of HRD. SBS3 was more strongly associated with TTPp in patients with GI malignancies and may be complementary to using HRD and BRCA status in identifying patients who benefit from platinum therapy.

Integrative analysis of KRAS wildtype metastatic pancreatic ductal adenocarcinoma reveals mutation and expression-based similarities to cholangiocarcinoma.

Oncogenic KRAS mutations are absent in approximately 10% of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) and may represent a subgroup of mPDAC with therapeutic options beyond standard-of-care cytotoxic chemotherapy. While distinct gene fusions have been implicated in KRAS wildtype mPDAC, information regarding other types of mutations remain limited, and gene expression patterns associated with KRAS wildtype mPDAC have not been reported. Here, we leverage sequencing data from the PanGen trial to perform comprehensive characterization of the molecular landscape of KRAS wildtype mPDAC and reveal increased frequency of chr1q amplification encompassing transcription factors PROX1 and NR5A2. By leveraging data from colorectal adenocarcinoma and cholangiocarcinoma samples, we highlight similarities between cholangiocarcinoma and KRAS wildtype mPDAC involving both mutation and expression-based signatures and validate these findings using an independent dataset. These data further establish KRAS wildtype mPDAC as a unique molecular entity, with therapeutic opportunities extending beyond gene fusion events.

Whole-genome and transcriptome analysis of advanced adrenocortical cancer highlights multiple alterations affecting epigenome and DNA repair pathways.

Adrenocortical cancer (ACC) is a rare cancer of the adrenal gland. Several driver mutations have been identified in both primary and metastatic ACCs, but the therapeutic options are still limited. We performed whole-genome and transcriptome sequencing on seven patients with metastatic ACC. Integrative analysis of mutations, RNA expression changes, mutation signature, and homologous recombination deficiency (HRD) analysis was performed. Mutations affecting CTNNB1 and TP53 and frequent loss of heterozygosity (LOH) events were observed in our cohort. Alterations affecting genes involved in cell cycle (RB1, CDKN2A, CDKN2B), DNA repair pathways (MUTYH, BRCA2, ATM, RAD52, MLH1, MSH6), and telomere maintenance (TERF2 and TERT) consisting of somatic and germline mutations, structural variants, and expression outliers were also observed. HRDetect, which aggregates six HRD-associated mutation signatures, identified a subset of cases as HRD. Genomic alterations affecting genes involved in epigenetic regulation were also identified, including structural variants (SWI/SNF genes and histone methyltransferases), and copy gains and concurrent high expression of KDM5A, which may contribute to epigenomic deregulation. Findings from this study highlight HRD and epigenomic pathways as potential therapeutic targets and suggest a subgroup of patients may benefit from a diverse array of molecularly targeted therapies in ACC, a rare disease in urgent need of therapeutic strategies.

Subtype-Discordant Pancreatic Ductal Adenocarcinoma Tumors Show Intermediate Clinical and Molecular Characteristics.

PURPOSE: RNA-sequencing-based subtyping of pancreatic ductal adenocarcinoma (PDAC) has been reported by multiple research groups, each using different methodologies and patient cohorts. "Classical" and "basal-like" PDAC subtypes are associated with survival differences, with basal-like tumors associated with worse prognosis. We amalgamated various PDAC subtyping tools to evaluate the potential of such tools to be reliable in clinical practice. EXPERIMENTAL DESIGN: Sequencing data for 574 PDAC tumors was obtained from prospective trials and retrospective public databases. Six published PDAC subtyping strategies (Moffitt regression tools, clustering-based Moffitt, Collisson, Bailey, and Karasinska subtypes) were used on each sample, and results were tested for subtype call consistency and association with survival. RESULTS: Basal-like and classical subtype calls were concordant in 88% of patient samples, and survival outcomes were significantly different (P < 0.05) between prognostic subtypes. Twelve percent of tumors had subtype-discordant calls across the different methods, showing intermediate survival in univariate and multivariate survival analyses. Transcriptional profiles compatible with that of a hybrid subtype signature were observed for subtype-discordant tumors, in which classical and basal-like genes were concomitantly expressed. Subtype-discordant tumors showed intermediate molecular characteristics, including subtyping gene expression (P < 0.0001) and mutant KRAS allelic imbalance (P < 0.001). CONCLUSIONS: Nearly 1 in 6 patients with PDAC have tumors that fail to reliably fall into the classical or basal-like PDAC subtype categories, based on two regression tools aimed toward clinical practice. Rather, these patient tumors show intermediate prognostic and molecular traits. We propose close consideration of the non-binary nature of PDAC subtypes for future incorporation of subtyping into clinical practice.

Pan-cancer analysis of advanced patient tumors reveals interactions between therapy and genomic landscapes.

Advanced and metastatic tumors with complex treatment histories drive cancer mortality. Here we describe the POG570 cohort, a comprehensive whole-genome, transcriptome and clinical dataset, amenable for exploration of the impacts of therapies on genomic landscapes. Previous exposure to DNA-damaging chemotherapies and mutations affecting DNA repair genes, including POLQ and genes encoding Polζ, were associated with genome-wide, therapy-induced mutagenesis. Exposure to platinum therapies coincided with signatures SBS31 and DSB5 and, when combined with DNA synthesis inhibitors, signature SBS17b. Alterations in ESR1, EGFR, CTNNB1, FGFR1, VEGFA and DPYD were consistent with drug resistance and sensitivity. Recurrent noncoding events were found in regulatory region hotspots of genes including TERT, PLEKHS1, AP2A1 and ADGRG6. Mutation burden and immune signatures corresponded with overall survival and response to immunotherapy. Our data offer a rich resource for investigation of advanced cancers and interpretation of whole-genome and transcriptome sequencing in the context of a cancer clinic.

Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding.

The crucial role of Myc as an oncoprotein and as a key regulator of cell growth makes it essential to understand the molecular basis of Myc function. The N-terminal region of c-Myc coordinates a wealth of protein interactions involved in transformation, differentiation and apoptosis. We have characterized in detail the intrinsically disordered properties of Myc-1-88, where hierarchical phosphorylation of S62 and T58 regulates activation and destruction of the Myc protein. By nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we show that although Myc occupies a very heterogeneous conformational space, we find transiently structured regions in residues 22-33 and in the Myc homology box I (MBI; residues 45-65); both these regions are conserved in other members of the Myc family. Binding of Bin1 to Myc-1-88 as assayed by NMR and surface plasmon resonance (SPR) revealed primary binding to the S62 region in a dynamically disordered and multivalent complex, accompanied by population shifts leading to altered intramolecular conformational dynamics. These findings expand the increasingly recognized concept of intrinsically disordered regions mediating transient interactions to Myc, a key transcriptional regulator of major medical importance, and have important implications for further understanding its multifaceted role in gene regulation.

H-start for exclusively heteronuclear NMR spectroscopy: the case of intrinsically disordered proteins.

Here, we present a series of exclusively heteronuclear multidimensional NMR experiments, based on 13C direct detection, which exploit the (1)H polarization as a starting source to increase the signal-to-noise ratio. This contributes to make this spectroscopy more useful and usable. Examples are reported for a suitable system such as securin, an intrinsically disordered protein of 22 kDa.

Structural and dynamic characterization of intrinsically disordered human securin by NMR spectroscopy.

Understanding the molecular action of securin, the inhibitor of separase in mitosis, is of immense theoretical and biomedical importance. The residue-level structural description of an intrinsically disordered protein of this length (202 amino acids, containing 24 prolines), however, represents a particular challenge. Here we combined (1)H-detected and (13)C-detected protonless NMR experiments to achieve full assignment of securin's backbone amide resonances. Chemical shifts, (15)N relaxation rates (R(1), R(2), (1)H-(15)N NOEs), (1)H exchange rates with the solvent (CLEANEX-PM), and (1)H-(15)N residual dipolar couplings were determined along the entire length of the protein. This analysis showed that securin is not entirely disordered, but segregates into a largely disordered N-terminal half and a C-terminal half with transient segmental order, within which the segment D(150)-F(159) has a significant helical tendency and segments E(113)-S(127) and W(174)-L(178) also show a significant deviation from random-coil behavior. These results, in combination with bioinformatic and biochemical data on the securin/separase interaction, shed light on the inhibitory action of securin on separase.

Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2.

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state (1)H NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.e., subdomains A, B, and C in CSD1 and the proline-rich region (PRR) in MAP2c, that are destined to form contacts with their targets. For CSD1, nonadditivity of the CD spectra of its two halves and suboptimal hydration of the full-length protein measured by solid-state NMR demonstrate that long-range tertiary interactions provide the structural background of this structural feature. In MAP2c, such tertiary interactions are absent, which points to the importance of local structural constraints. In fact, urea and temperature dependence of the CD spectrum of its PRR reveals the presence of the extended and rather stiff polyproline II helix conformation that keeps the interaction site exposed. These data suggest that functionally significant residual structure exists in both of these IUPs. This structure, manifest as either transient local and/or global organization, ensures the spatial exposure of short contact segments on the surface. Pertinent data from other IUPs suggest that the presence of such recognition motifs may be a general feature of disordered proteins. To emphasize the possible importance of this structural trait, we propose that these motifs be called primary contact sites in IUPs.