Precision Oncology in Canada: Converting Vision to Reality with Lessons from International Programs.

Canada's healthcare system, like others worldwide, is immersed in a process of evolution, attempting to adapt conventional frameworks of health technology assessment (HTA) and funding models to a new landscape of precision medicine in oncology. In particular, the need for real-world evidence in Canada is not matched by the necessary infrastructure and technologies required to integrate genomic and clinical data. Since healthcare systems in many developed nations face similar challenges, we adopted a solutions-based approach and conducted a search of worldwide programs in personalized medicine, with an emphasis on precision oncology. This search strategy included review articles published between 1 January 2016 and 1 March 2021 and hand-searches of their reference lists for relevant publications back to 1 December 2005. Thirty-nine initiatives across 37 countries in Europe, Australasia, Africa, and the Americas had the potential to lead to real-world data (RWD) on the clinical utility of oncology biomarkers. We highlight four initiatives with helpful lessons for Canada: Genomic Medicine France 2025, UNICANCER, the German Medical Informatics Initiative, and CANCER-ID. Among the 35 other programs evaluated, the main themes included the need for collaboration and systems to support data harmonization across multiple jurisdictions. In order to generate RWD in precision oncology that will prove acceptable to HTA bodies, Canada must take a national approach to biomarker strategy and unite all stakeholders at the highest level to overcome jurisdictional and technological barriers.

MATE2 Expression Is Associated with Cancer Cell Response to Metformin.

BACKGROUND: There is great interest in repurposing the commonly prescribed anti-diabetic drug metformin for cancer therapy. Intracellular uptake and retention of metformin is affected by the expression of organic cation transporters (OCT) 1-3 and by multidrug and toxic compound extrusion (MATE) 1-2. Inside cells, metformin inhibits mitochondrial function, which leads to reduced oxygen consumption and inhibition of proliferation. Reduced oxygen consumption can lead to improved tumor oxygenation and radiation response. PURPOSE: Here we sought to determine if there is an association between the effects of metformin on inhibiting oxygen consumption, proliferation and expression of OCTs and MATEs in a panel of 19 cancer cell lines. RESULTS: There was relatively large variability in the anti-proliferative response of different cell lines to metformin, with a subset of cell lines being very resistant. In contrast, all cell lines demonstrated sensitivity to the inhibition of oxygen consumption by metformin, with relatively small variation. The expression of OCT1 correlated with expression of both OCT2 and OCT3. OCT1 and OCT2 were relatively uniformly expressed, whereas expression of OCT3, MATE1 and MATE2 showed substantial variation across lines. There were statistically significant associations between resistance to inhibition of proliferation and MATE2 expression, as well as between sensitivity to inhibition of oxygen consumption and OCT3 expression. One cell line (LNCaP) with high OCT3 and low MATE2 expression in concert, had substantially higher intracellular metformin concentration than other cell lines, and was exquisitely sensitive to both anti-proliferative and anti-respiratory effects. In all other cell lines, the concentration of metformin required to inhibit oxygen consumption acutely in vitro was substantially higher than that achieved in the plasma of diabetic patients. However, administering anti-diabetic doses of metformin to tumor-bearing mice resulted in intratumoral accumulation of metformin and reduced hypoxic tumor fractions. CONCLUSIONS: All cancer cells are susceptible to inhibition of oxygen consumption by metformin, which results in reduced hypoxic tumor fractions beneficial for the response to radiotherapy. High MATE2 expression may result in resistance to the anti-proliferative effect of metformin and should be considered as a negative predictive biomarker in clinical trials.

Mitochondrial DNA damage by bleomycin induces AML cell death.

Mitochondria contain multiple copies of their own 16.6 kb circular genome. To explore the impact of mitochondrial DNA (mtDNA) damage on mitochondrial (mt) function and viability of AML cells, we screened a panel of DNA damaging chemotherapeutic agents to identify drugs that could damage mtDNA. We identified bleomycin as an agent that damaged mtDNA in AML cells at concentrations that induced cell death. Bleomycin also induced mtDNA damage in primary AML samples. Consistent with the observed mtDNA damage, bleomycin reduced mt mass and basal oxygen consumption in AML cells. We also demonstrated that the observed mtDNA damage was functionally important for bleomycin-induced cell death. Finally, bleomycin delayed tumor growth in xenograft mouse models of AML and anti-leukemic concentrations of the drug induced mtDNA damage in AML cells preferentially over normal lung tissue. Taken together, mtDNA-targeted therapy may be an effective strategy to target AML cells and bleomycin could be useful in the treatment of this disease.

Srs2 enables checkpoint recovery by promoting disassembly of DNA damage foci from chromatin.

Following DNA repair, checkpoint signalling must be abated to resume cell cycling in a phenomenon known as checkpoint recovery. Although a number of genes have been implicated in the recovery process, it is still unknown whether checkpoint recovery is caused by a signalling network activated by DNA repair or whether it is the result of the loss of DNA structures that elicit the checkpoint. Here we show that checkpoint recovery can be uncoupled from bulk chromosome DNA repair if single-stranded (ss) DNA persists. This situation occurs in cells that are deficient in the Srs2 helicase, a protein that antagonizes Rad51. We report that srs2Δ cells fail to eliminate Ddc2 and RPA subnuclear foci following bulk chromosome repair due to the persistence of ssDNA. In contrast to cells with DNA double-strand breaks that remain unrepaired, srs2Δ cells remove the 9-1-1 checkpoint clamp from chromatin after repair. However, despite the loss of the 9-1-1 clamp, Dpb11 remains associated with chromatin to promote checkpoint activity. Our work indicates that Srs2 promotes checkpoint recovery by removing Rad51 after DNA repair. A failure to remove Rad51 causes persistence of ssDNA and the checkpoint signal. Therefore, we conclude that cells initiate recovery when the DNA structures that elicit the checkpoint are eliminated.

Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX.

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, gamma-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high-resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

Engineering a DNA damage response without DNA damage.

Recent work has achieved the feat of activating the DNA damage checkpoint in the absence of DNA damage, revealing the importance of protein-chromatin associations for the activation, amplification and maintenance of the DNA damage response.

Rad6-Rad18 mediates a eukaryotic SOS response by ubiquitinating the 9-1-1 checkpoint clamp.

Bacteria employ a coordinated SOS response to DNA damage by enhancing transcription, translesion synthesis, and recombination; a similar phenomenon has not been reported in eukaryotes. Here, we demonstrate that the ubiquitination complex Rad6-Rad18 is required for the increased transcription of a large number of yeast genes in response to DNA damage. Rad6-Rad18 promotes DNA-damage-dependent transcriptional induction as well as checkpoint functions by catalyzing monoubiquitination at the K197 residue of the Rad17 subunit of the 9-1-1 complex. Rad17 ubiquitination invokes both DNA damage responsive pathways by promoting efficient Rad53 phosphorylation, possibly through the recruitment or maintenance of the 9-1-1 clamp at sites of lesions. Taken together, the Rad6-Rad18 complex is involved in the control of global gene regulation in a way reminiscent of the bacterial SOS response and plays key roles in coordinating several DNA damage response pathways through ubiquitination of two DNA clamps, PCNA and 9-1-1.

Condensin is required for chromosome arm cohesion during mitosis.

We describe a novel requirement for the condensin complex in sister chromatid cohesion in Saccharomyces cerevisiae. Strikingly, condensin-dependent cohesion can be distinguished from cohesin-based pairing by a number of criteria. First, condensin is required to maintain cohesion at several chromosomal arm sites but, in contrast to cohesin, is not required at either centromere or telomere-proximal loci. Second, condensin-dependent interlinks are established during mitosis independently of DNA replication and are reversible within a single cell cycle. Third, the loss of condensin-dependent linkages occurs without affecting cohesin levels at the separated URA3 locus. We propose that, during mitosis, robust sister chromatid cohesion along chromosome arms requires both condensinand cohesin-dependent mechanisms, which function independently of each other. We discuss the implications of our results for current models of sister chromatid cohesion.