The Roles of the Saccharomyces cerevisiae RecQ Helicase SGS1 in Meiotic Genome Surveillance

leads to an increase in synapsis initiation complexes and axial associations leading to the proposal that it has an early role in unwinding surplus strand invasion events. Physical studies of recombination intermediates implicate it in the dissolution of double Holliday junctions between sister chromatids. chromosomes may sometimes remain entangled at the end of pre-meiotic replication. This, combined with reciprocal crossing over, could lead to physical destruction of the recombined and entangled chromosomes. We hypothesise that Sgs1, acting in concert with the topoisomerase Top2, resolves these structures.This work provides evidence that Sgs1 interacts with various partner proteins to maintain genome stability throughout meiosis

Investigating the role of Sgs1 in the suppression of meiotic homeologous recombination in Saccharomyces cerevisiae

Homeologous recombination is the genetic exchange that occurs between diverged DNA sequences. This type of recombination can be detrimental to the cell, as it could lead to deletions, duplications and even chromosome loss. Therefore, it must be suppressed in order to maintain the integrity of the genome. The mismatch repair (MMR) complex, along with the 3’-to-5’ helicase Sgs1, has been implicated in this early role in meiosis. We propose a model in which the MMR complex scans the genome searching for single end invasion (SEI) events occurring between diverged sequences. On finding such events, the MMR complex binds to them and impedes their progression. The MMR complex then recruits the Sgs1 helicase which unwinds the heteroduplex DNA, allowing the invading strand to continue its homology search. To test this hypothesis, we investigated whether the interaction between Sgs1 and one member of this MMR complex, Mlh1, affected the ability of Sgs1 to suppress homeologous recombination in the baker’s yeast Saccharomyces cerevisiae. In addition, we investigated which domains of Sgs1 were required for this suppression. The data presented here show that the interaction between Sgs1 and Mlh1 may be important in the suppression of homeologous recombination at the SEI stage. In addition to this, we present data suggesting that the interaction between Sgs1 and the type IA topoisomerase Top3 may also be important in the resolution of recombination intermediates formed between diverged sequences. We suggest that there may still be additional factors that are utilised by the cell in order to maintain the barrier to inter-species recombination. Sgs1 has also been suggested to function at a later stage of meiosis, in the decatenation of Holliday junctions. This process was proposed to be carried out in concert with Top3. We present data that conflict with this hypothesis. We propose that interactions between Sgs1 and the type II topoisomerase Top2 are required in the decatenation of pre-existing replication errors prior to the onset of meiosis. The data implicate Sgs1 in the pre-meiotic replication checkpoint to aid in the repair of errors caused during DNA replication prior to meiosis. We also hypothesise an additional role of Sgs1 in the activation of this pre-meiotic replication checkpoint for the process of sporulation. This investigation therefore emphasises the importance of Sgs1 in the early stages of meiosis

Investigating the role of Sgs1 in the suppression of meiotic homeologous recombination in

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Induced Dependence On The ALK Kinase Inhibitor Crizotinib In Formerly Sensitive Anaplastic Large Cell Lymphoma Cells

Abstract Kinase inhibitors can be highly effective against cancers driven by specific oncogenic kinase proteins, but resistance usually develops after prolonged treatment. Target-dependent mechanisms, typically acquired second-site mutations that prevent drug binding, or target-independent mechanisms, such as downstream or parallel pathway activation, may mediate resistance. Prolonged exposure of kinase-dependent cell lines to inhibitors in vitro to select for resistant populations has identified clinically relevant resistance mechanisms against a number of inhibitors. ALK-positive anaplastic large-cell lymphoma (ALCL) is an uncommon form of peripheral T-cell lymphoma (PTCL) driven by the NPM-ALK fusion kinase, resulting from a t(2;5)(p23;q35) chromosomal rearrangement. ALK inhibitors developed for lung cancer, where a different ALK fusion (EML4-ALK) is found in 6% of cases, have shown promise clinically as a new treatment modality for ALK+ ALCL patients. In this study, we selected three NPM-ALK-dependent ALCL cell lines (Karpas-299, SUP-M2, and SU-DHL-1) for resistance to the ALK inhibitor crizotinib. All three lines initially were highly sensitive the drug (IC50 1 µM (Karpas-299CR1000). This line had greatly increased viability in crizotinib at concentrations up to 1 µM, compared to untreated cells, suggesting the drug actually maintained growth. Plated in 250 nM crizotinib, Karpas-299CR1000 grows rapidly, similar to the parent line growing in drug-free media. Plated in drug-free media, however, the line’s viability drops rapidly to zero and dies, similar to the parent line plated in 250 nM crizotinib. Therefore the drug that once shut off the signaling necessary for the cells’ survival, after prolonged exposure (>9 months), became necessary to maintain it in the sub-clonal population. Melanoma cells driven by BRAF-V600E selected for vemurafenib resistance had a similar phenotype in a recent report (Das Thakur, et al., 2013). These cells amplified mutant BRAF to overcome the inhibitor but came to require the drug to counteract BRAF over-activity with amplification beyond a certain point. Preliminarily, we find a similar effect of NPM-ALK amplification in Karpas-299CR1000 cells but are actively exploring mechanism. In sum, our study shows that long-term exposure of ALK+ ALCL cells to an ALK inhibitor may induce a resistant phenotype that comes to depend on drug presence to prevent over-activity of the mutant kinase. Disclosures: No relevant conflicts of interest to declare

Abstract 5451: Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing

Abstract Activated Anaplastic Lymphoma Kinase (ALK) is seen in several cancers, including 2-7% of non-small cell lung cancer and ∼ 70% of Anaplastic Large Cell Lymphoma (ALCL). The most common ALK fusion in ALCL is a translocation, t(2;5)(p23;35), placing the kinase domain of ALK before the constitutively active promoter of nucleophosmin (NPM). Activated ALK drives oncogenesis by turning on multiple proliferative pathways. The success of tyrosine kinase inhibitors (TKIs) in ALK+ lung cancer has prompted evaluation of their use in ALK+ ALCL as a therapeutic strategy for patients who fail combination chemotherapy. Clinical studies in lung cancer show resistance to these drugs limits progression-free survival. We aim to identify resistance mechanisms against approved TKIs, crizotinib and ceritinib, in ALK+ ALCL. We selected for resistance in 3 patient-derived cell lines through propagation in gradually increasing drug concentrations. We assessed resistant subclones with long-insert whole-genome sequencing, viability and proliferation assays, qPCR and western blots. Similar profiling was carried out upon drug withdrawal. We validated our findings in cytokine-dependent murine pro-B cells transformed with NPM-ALK during ceritinib incubation. We xenografted resistant clones to SCID mice and assessed tumour burden over time. Resistant clones showed viability stimulation by TKIs and when washed out of drug, these clones underwent apoptosis due to hyper-stimulation of ALK signalling. Genomic amplification of NPM-ALK was seen leading to an increased expression at the mRNA and protein levels. TKI-dependence therefore was co-selected with resistance, showing for the first time toxicity to cancer cells due to an overdose of ALK signalling. We validated our findings in IL3-dependent FL/5.12 cells, which also showed co-selection for drug resistance and dependence due to ALK over-expression. Resistant ALCL lines selected for ability to grow without TKI behaved similarly to parent lines, showing no stimulation of viability in presence of drug. ALK mRNA and protein also return to baseline in these cells. SCID mice injected with resistant cells required TKI treatment for tumour engraftment and tumour burden decreased when drug dosing was stopped. When tumours re-grew, drug treatment was reinitiated, generating a second response demonstrating in vivo that NPM-ALK up-regulation results in both resistance and dependence on the TKI and resistant cells die from an overdose of ALK signalling in the absence of drug. Our results reveal that up-regulation of the fusion-ALK drug target is a resistance mechanism, previously un-reported in any ALK+ cancers. Up-regulation of NPM-ALK provides cells means to acquire resistance but also results in ALK overdose when drug is withdrawn, revealing intermittent dosing as a potential therapeutic strategy to prolong tumour control in ALK + patients. Citation Format: Soumya Sundara Rajan, Amit Dipak Amin, Matthew Groysman, Praechompoo Pongtornpipat, Jonathan Schatz. Over-expression of NPM-ALK drives resistance to TKIs in ALK+ ALCL but is toxic upon drug withdrawal, permitting prolonged tumour control through discontinuous dosing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2015-545

Oncogene Overdose: Too Much of a Bad Thing for Oncogene-Addicted Cancer Cells

Acquired resistance to targeted inhibitors remains a major, and inevitable, obstacle in the treatment of oncogene-addicted cancers. Newer-generation inhibitors may help overcome resistance mutations, and inhibitor combinations can target parallel pathways, but durable benefit to patients remains elusive in most clinical scenarios. Now, recent studies suggest a third approach may be available in some cases—exploitation of oncogene overexpression that may arise to promote resistance. Here, we discuss the importance of maintaining oncogenic signaling at “just-right” levels in cells, with too much signaling, or oncogene overdose, being potentially as detrimental as too little. This is highlighted in particular by recent studies of mutant-BRAF in melanoma and the fusion kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in anaplastic large cell lymphoma. Oncogene overdose may be exploitable to prolong tumor control through intermittent dosing in some cases, and studies of acute lymphoid leukemias suggest that it may be specifically pharmacologically inducible

CRISPR/Cas9 Generation of Npm1-Alk in Transplantable Murine Hematopoietic Stem Cells Accurately Models ALK-Positive Lymphoma in Recipients

Abstract Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies. Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3' ALK kinase domain to the 5' oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients. Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients. We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. Disclosures No relevant conflicts of interest to declare

Diffuse large B-cell lymphoma: can genomics improve treatment options for a curable cancer?

Gene-expression profiling and next-generation sequencing have defined diffuse large B-cell lymphoma (DLBCL), the most common lymphoma diagnosis, as a heterogeneous group of subentities. Despite ongoing explosions of data illuminating disparate pathogenic mechanisms, however, the five-drug chemoimmunotherapy combination R-CHOP remains the frontline standard treatment. This has not changed in 15 years, since the anti-CD20 monoclonal antibody rituximab was added to the CHOP backbone, which first entered use in the 1970s. At least a third of patients are not cured by R-CHOP, and relapsed or refractory DLBCL is fatal in ∼90%. Targeted small-molecule inhibitors against distinct molecular pathways activated in different subgroups of DLBCL have so far translated poorly into the clinic, justifying the ongoing reliance on R-CHOP and other long-established chemotherapy-driven combinations. New drugs and improved identification of biomarkers in real time, however, show potential to change the situation eventually, despite some recent setbacks. Here, we review established and putative molecular drivers of DLBCL identified through large-scale genomics, highlighting among other things the care that must be taken when differentiating drivers from passengers, which is influenced by the promiscuity of activation-induced cytidine deaminase. Furthermore, we discuss why, despite having so much genomic data available, it has been difficult to move toward personalized medicine for this umbrella disorder and some steps that may be taken to hasten the process