Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia.

In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Molecular characterisation of pancreatic ductal adenocarcinoma with NTRK fusions and review of the literature.

AIMS: The majority of pancreatic ductal adenocarcinomas (PDACs) harbour oncogenic mutations in KRAS with variants in TP53, CDKN2A and SMAD4 also prevalent. The presence of oncogenic fusions including NTRK fusions are rare but important to identify. Here we ascertain the prevalence of NTRK fusions and document their genomic characteristics in a large series of PDAC. METHODS: Whole genome sequencing and RNAseq were performed on a series of patients with resected or locally advanced/metastatic PDAC collected between 2008 and 2020 at a single institution. A subset of specimens underwent immunohistochemistry (IHC) analysis. Clinical and molecular characterisation and IHC sensitivity and specificity were evaluated. RESULTS: 400 patients were included (resected n=167; locally advanced/metastatic n=233). Three patients were identified as harbouring an NTRK fusion, two EML4-NTRK3 (KRAS-WT) and a single novel KANK1-NTRK3 fusion. The latter occurring in the presence of a subclonal KRAS mutation. Typical PDAC drivers were present including mutations in TP53 and CDKN2A. Substitution base signatures and tumour mutational burden were similar to typical PDAC. The prevalence of NTRK fusions was 0.8% (3/400), while in KRAS wild-type tumours, it was 6.25% (2/32). DNA prediction alone documented six false-positive cases. RNA analysis correctly identified the in-frame fusion transcripts. IHC analysis was negative in the KANK1-NTRK3 fusion but positive in a EML4-NTRK3 case, highlighting lower sensitivity of IHC. CONCLUSION: NTRK fusions are rare; however, with emerging therapeutic options targeting these fusions, detection is vital. Reflex testing for KRAS mutations and subsequent RNA-based screening could help identify these cases in PDAC.

Author Correction: Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution.

Pancreatic adenocarcinoma presents as a spectrum of a highly aggressive disease in patients. The basis of this disease heterogeneity has proved difficult to resolve due to poor tumor cellularity and extensive genomic instability. To address this, a dataset of whole genomes and transcriptomes was generated from purified epithelium of primary and metastatic tumors. Transcriptome analysis demonstrated that molecular subtypes are a product of a gene expression continuum driven by a mixture of intratumoral subpopulations, which was confirmed by single-cell analysis. Integrated whole-genome analysis uncovered that molecular subtypes are linked to specific copy number aberrations in genes such as mutant KRAS and GATA6. By mapping tumor genetic histories, tetraploidization emerged as a key mutational process behind these events. Taken together, these data support the premise that the constellation of genomic aberrations in the tumor gives rise to the molecular subtype, and that disease heterogeneity is due to ongoing genomic instability during progression.

A renewed model of pancreatic cancer evolution based on genomic rearrangement patterns.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

Truncating Erythropoietin Receptor Rearrangements in Acute Lymphoblastic Leukemia.

Chromosomal rearrangements are a hallmark of acute lymphoblastic leukemia (ALL) and are important ALL initiating events. We describe four different rearrangements of the erythropoietin receptor gene EPOR in Philadelphia chromosome-like (Ph-like) ALL. All of these rearrangements result in truncation of the cytoplasmic tail of EPOR at residues similar to those mutated in primary familial congenital polycythemia, with preservation of the proximal tyrosine essential for receptor activation and loss of distal regulatory residues. This resulted in deregulated EPOR expression, hypersensitivity to erythropoietin stimulation, and heightened JAK-STAT activation. Expression of truncated EPOR in mouse B cell progenitors induced ALL in vivo. Human leukemic cells with EPOR rearrangements were sensitive to JAK-STAT inhibition, suggesting a therapeutic option in high-risk ALL.

Late onset of symptoms in an atypical patient with the cblJ inborn error of vitamin B12 metabolism: diagnosis and novel mutation revealed by exome sequencing.

Inborn errors of vitamin B(12) (cobalamin) metabolism are characterized by decreased production of active cobalamin cofactors and subsequent deficiencies in the activities of methionine synthase and methylmalonyl-CoA mutase. With the recent discovery of the cblJ defect in two patients with phenotypes mimicking the cblF defect, there are nine genes known to be involved in cobalamin metabolism. The new defect is caused by mutations in the ABCD4 gene, encoding an ABC transporter. At the moment, there is no clear distinction between the cblJ and cblF defects either clinically or biochemically, and both defects result in blocks in the transport of cobalamin from the lysosome to the cytoplasm. A patient was diagnosed with hyperhomocysteinemia and methylmalonic aciduria at the age of 8 years. Incorporations of both [(14)C]propionate and [(14)C]methyltetrahydrofolate in cultured fibroblasts were within reference ranges and thus too high to allow for complementation analysis. We observed decreased synthesis of both adenosylcobalamin and methylcobalamin and accumulation of unmetabolized cyanocobalamin. Exome sequencing was performed to identify causative mutation(s) and Sanger re-sequencing was performed to validate segregation of mutation in the family. By this approach, a homozygous mutation, c.423C>G, in the ABCD4 gene was identified. Here, we report the successful application of exome sequencing for diagnosis of a rare inborn error of vitamin B(12) metabolism in a patient whose unusual presentation precluded diagnosis using standard biochemical and genetic approaches. The patient represents only the third known patient with the cblJ disorder.

Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.