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Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype., Author summary Cancer is a genetic disease caused by mutations that lead to uncontrolled cell proliferation and other tumour properties. Defects in DNA repair or replication can lead to cancer development by increasing the likelihood that cancer-causing mutations will happen. Here we look at a pathogenic variant of a polymerase involved in genome replication (DNA polymerase ε POLE-P286R). This variant is associated with highly mutated cancer genomes. By introducing this mutation into the polymerase ε gene of a model organism, fission yeast, we show that it causes a large increase in single base substitutions, scattered throughout the genome. The sequence context of mutations is similar in fission yeast and humans, suggesting that the yeast model is useful for understanding how POLE-P286R causes such a high mutation rate. Yeast POLE-P286R cells show slow chromosome replication, suggesting that the polymerase has difficulty in copying certain chromosomal regions. Yeast POLE-P286R cells become inviable when the concentration of dNTP building blocks for DNA synthesis is increased, probably because the mutation rate is pushed to an intolerable level. Interestingly, we find that specialised polymerases that are tolerant of DNA damage contribute to the high mutation rate caused by POLE-P286R. These findings have implications for the therapy of POLE-P286R tumours.

Somatic mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, microsatellite stability and a unique mutational signature, with a predominance of C > A transversions in the context TCT. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few frameshift mutations. The mutations are equally distributed across different genomic regions, but they occur within an AT-rich context. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of persistent ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.AUTHOR SUMMARYCancer is a genetic disease caused by mutations that lead to uncontrolled cell proliferation and other tumour properties. Defects in DNA repair or replication can lead to cancer development by increasing the likelihood that cancer-causing mutations will happen. Here we look at a pathogenic variant of a polymerase involved in genome replication (DNA polymerase ⍰⍰⍰POLE-P286R). This variant is associated with highly mutated cancer genomes. By introducing this mutation into the polymerase ⍰ gene of a model organism, fission yeast, we show that it causes a large increase in single base substitutions, scattered throughout the genome. The sequence context of mutations is similar in fission yeast and humans, suggesting that the yeast model is useful for understanding how POLE-P286R causes such a high mutation rate. Yeast POLE-P286R cells show slow chromosome replication, suggesting that the polymerase has difficulty in copying certain chromosomal regions. Yeast POLE-P286R cells become inviable when the concentration of dNTP building blocks for DNA synthesis is increased, probably because the mutation rate is pushed to an intolerable level. Interestingly, we find that specialised polymerases that are tolerant of DNA damage contribute to the high mutation rate caused by POLE-P286R. These findings have implications for the therapy of POLE-P286R tumours.

The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.

Background Bromodomain and extra-terminal (BET) proteins, including the ubiquitous BRD2/3/4 proteins, are epigenetic readers implicated in c-MYC transcription, cellular proliferation, cell-cycle progression, RNA elongation and DNA damage response. Using shRNA screening and BRD inhibitors, BRD4 has been established as a promising therapeutic target in acute leukemia (Zuber, Nature 2011). In the present study, we investigated the in vitro anti-leukemic effects of the small-molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland). Methods Expression of BRD2/3/4 and c-MYC was assessed by RQ-PCR in 5 myeloid (HL60, KG1, KG1a, K562, NOMO1) and 4 lymphoid (Jurkat, RS4-11, BV173, TOM1) leukemia cell lines and by Western blotting (WB) using commercial antibodies in the HL60, K562, Jurkat and RS4-11 lines. Nineteen AML and ten ALL patient banked leukemic cells were assayed by RQ-PCR only. Cell viability and IC50 values were assessed in cell lines by MTT assays after exposure to OTX015 (0.1nM-10µM) for 72h. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Induction of apoptosis was evaluated in cell lines and patient cells by outer membrane phosphatidylserine exposure and PI incorporation at 72 hours with increasing doses of OTX015 (25nM-500nM). Caspase-3 activation and mitochondrial cytochrome c release were studied by immunofluorescence (IF). Maturation was assessed by morphological studies after MGG staining and detection of CD11b by FACS analysis. Modulation of BRD2/3/4 proteins was investigated by WB. Results OTX015 IC50 values were in the submicromolar range for KG1 and the MLL-driven NOMO1 cell lines (198.3 and 229.1nM, respectively), while K562 was the most resistant myeloid line, with an IC50 of 11.3µM. In contrast, in lymphoid cell lines tested, IC50 values ranged from 34.2 to 249.7nM, with the MLL-driven cell line RS4-11 being the most sensitive. Cell cycle arrest in subG1/G1 to S transition was observed in 8/9 cell lines and was most pronounced in RS4-11 and BV173. Significant apoptosis (up to 88% Annexin V positive cells) was only observed in KG1a and NOMO1 among myeloid cell lines, while OTX015 induced apoptosis in all lymphoid cell lines tested, ranging from 57% in RS4-11 to 90% in the BCR-ABL+ TOM1 cells. Similarly, OTX015 triggered caspase-dependent cell death, as NOMO1 and RS4-11 displayed significant caspase-3 activation and cytochrome c release, when compared to the resistant K562 cell line. Seven primary patient fresh samples (5 AML, 2ALL) were also analyzed. Ex vivo treatment induced apoptosis ranged from 35% to 87% in 6/7 patients. Exposure to OTX015 at 500nM for 7 days induced maturation in 51% and 65% of HL60 cells as detected by CD11b expression and morphology, respectively. Baseline expression of BRD2/3/4 varied among cell lines or patient samples, lower BRD2/3/4 expression levels were observed in the BCR-ABL+ K562 and BV173 cell lines, as well as in the 4 BCR-ABL+ ALL samples analyzed. Upon OTX015 exposure, down-regulation of the BRD4 target gene c-MYC was observed in all cell lines, without clear correlation with the proliferation inhibition rate and/or the intensity of induced apoptosis while no consistent BRD2/3/4 mRNAs down-regulation was seen. Interestingly, BRD2 protein was down-regulated in HL60, Jurkat and RS4-11 cell lines, but not in the K562 cell line. Conclusion OTX015 affects cell viability, induces cell cycle arrest in G1/S phase, and is able to induce significant apoptosis in leukemic cell lines and fresh AML and ALL samples at submicromolar drug concentrations. These concentrations were achieved in the serum of healthy volunteers after safe administration of the drug. With such characteristics, OTX015 appears to be an attractive anti-leukemic therapy, currently under early evaluation in a Phase Ib dose-escalation trial conducted in relapsed/refractory AML/ALL patients. Disclosures: Riveiro: Oncology Therapeutic Development: Employment. Herait:Oncoethix: Employment. Dombret:Oncoethix: Research Funding.