Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

DNA damage response defects in hematologic malignancies: mechanistic insights and therapeutic strategies.

ABSTRACT: The DNA damage response (DDR) encompasses the detection and repair of DNA lesions and is fundamental to the maintenance of genome integrity. Germ line DDR alterations underlie hereditary chromosome instability syndromes by promoting the acquisition of pathogenic structural variants in hematopoietic cells, resulting in increased predisposition to hematologic malignancies. Also frequent in hematologic malignancies are somatic mutations of DDR genes, typically arising from replication stress triggered by oncogene activation or deregulated tumor proliferation that provides a selective pressure for DDR loss. These defects impair homology-directed DNA repair or replication stress response, leading to an excessive reliance on error-prone DNA repair mechanisms that results in genomic instability and tumor progression. In hematologic malignancies, loss-of-function DDR alterations confer clonal growth advantage and adverse prognostic impact but may also provide therapeutic opportunities. Selective targeting of functional dependencies arising from these defects could achieve synthetic lethality, a therapeutic concept exemplified by inhibition of poly-(adenosine 5'-diphosphate ribose) polymerase or the ataxia telangiectasia and Rad 3 related-CHK1-WEE1 axis in malignancies harboring the BRCAness phenotype or genetic defects that increase replication stress. Furthermore, the role of DDR defects as a source of tumor immunogenicity, as well as their impact on the cross talk between DDR, inflammation, and tumor immunity are increasingly recognized, thus providing rationale for combining DDR modulation with immune modulation. The nature of the DDR-immune interface and the cellular vulnerabilities conferred by DDR defects may nonetheless be disease-specific and remain incompletely understood in many hematologic malignancies. Their comprehensive elucidation will be critical for optimizing therapeutic strategies to target DDR defects in these diseases.

SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

Correction: Kwok et al. Targeting the p53 Pathway in CLL: State of the Art and Future Perspectives. Cancers 2021, 13, 4681.

The authors wish to make the following corrections to their paper [1] [...].

Targeting the p53 Pathway in CLL: State of the Art and Future Perspectives.

The p53 pathway is a desirable therapeutic target, owing to its critical role in the maintenance of genome integrity. This is exemplified in chronic lymphocytic leukemia (CLL), one of the most common adult hematologic malignancies, in which functional loss of p53 arising from genomic aberrations are frequently associated with clonal evolution, disease progression, and therapeutic resistance, even in the contemporary era of CLL targeted therapy and immunotherapy. Targeting the 'undruggable' p53 pathway therefore arguably represents the holy grail of cancer research. In recent years, several strategies have been proposed to exploit p53 pathway defects for cancer treatment. Such strategies include upregulating wild-type p53, restoring tumor suppressive function in mutant p53, inducing synthetic lethality by targeting collateral genome maintenance pathways, and harnessing the immunogenicity of p53 pathway aberrations. In this review, we will examine the biological and clinical implications of p53 pathway defects, as well as our progress towards development of therapeutic approaches targeting the p53 pathway, specifically within the context of CLL. We will appraise the opportunities and pitfalls associated with these therapeutic strategies, and evaluate their place amongst the array of new biological therapies for CLL.

TLR9 expression in chronic lymphocytic leukemia identifies a promigratory subpopulation and novel therapeutic target.

Chronic lymphocytic leukemia (CLL) remains incurable despite B-cell receptor-targeted inhibitors revolutionizing treatment. This suggests that other signaling molecules are involved in disease escape mechanisms and resistance. Toll-like receptor 9 (TLR9) is a promising candidate that is activated by unmethylated cytosine guanine dinucleotide-DNA. Here, we show that plasma from patients with CLL contains significantly more unmethylated DNA than plasma from healthy control subjects (P < .0001) and that cell-free DNA levels correlate with the prognostic markers CD38, ß2-microglobulin, and lymphocyte doubling time. Furthermore, elevated cell-free DNA was associated with shorter time to first treatment (hazard ratio, 4.0; P = .003). We also show that TLR9 expression was associated with in vitro CLL cell migration (P < .001), and intracellular endosomal TLR9 strongly correlated with aberrant surface expression (sTLR9; r = 0.9). In addition, lymph node-derived CLL cells exhibited increased sTLR9 (P = .016), and RNA-sequencing of paired sTLR9hi and sTLR9lo CLL cells revealed differential transcription of genes involved in TLR signaling, adhesion, motility, and inflammation in sTLR9hi cells. Mechanistically, a TLR9 agonist, ODN2006, promoted CLL cell migration (P < .001) that was mediated by p65 NF-κB and STAT3 transcription factor activation. Importantly, autologous plasma induced the same effects, which were reversed by a TLR9 antagonist. Furthermore, high TLR9 expression promoted engraftment and rapid disease progression in a NOD/Shi-scid/IL-2Rγnull mouse xenograft model. Finally, we showed that dual targeting of TLR9 and Bruton's tyrosine kinase (BTK) was strongly synergistic (median combination index, 0.2 at half maximal effective dose), which highlights the distinct role for TLR9 signaling in CLL and the potential for combined targeting of TLR9 and BTK as a more effective treatment strategy in this incurable disease.

Aniline-containing derivatives of parthenolide: Synthesis and anti-chronic lymphocytic leukaemia activity.

Parthenolide exhibits anti-leukaemia activity, whilst its synthetic modification to impart improve drug-like properties, including 1,4-conjugate addition of primary and secondary amines, have previously been used, 1,4-addition of aniline derivatives to parthenolide has not been fully explored. A protocol for such additions to parthenolide is outlined herein. Reaction conditions were determined using tulipane as a model Michael acceptor. Subsequently, aniline-containing parthenolide derivatives were prepared under the optimised conditions and single crystal X-ray diffraction structures were resolved for three of the compounds synthesised. The synthesised derivatives, along with compounds resulting from a side reaction, were tested for their in vitro anti-leukaemia activity using the chronic lymphocytic leukaemia (CLL) MEC1 cell line. Computational studies with the 2RAM protein structure suggested that the activity of the derivatives was independent of their in silico ability to dock with the Cys38 residue of NF-κB.

Integrative analysis of spontaneous CLL regression highlights genetic and microenvironmental interdependency in CLL.

Spontaneous regression is a recognized phenomenon in chronic lymphocytic leukemia (CLL) but its biological basis remains unknown. We undertook a detailed investigation of the biological and clinical features of 20 spontaneous CLL regression cases incorporating phenotypic, functional, transcriptomic, and genomic studies at sequential time points. All spontaneously regressed tumors were IGHV-mutated with no restricted IGHV usage or B-cell receptor (BCR) stereotypy. They exhibited shortened telomeres similar to nonregressing CLL, indicating prior proliferation. They also displayed low Ki-67, CD49d, cell-surface immunoglobulin M (IgM) expression and IgM-signaling response but high CXCR4 expression, indicating low proliferative activity associated with poor migration to proliferation centers, with these features becoming increasingly marked during regression. Spontaneously regressed CLL displayed a transcriptome profile characterized by downregulation of metabolic processes as well as MYC and its downstream targets compared with nonregressing CLL. Moreover, spontaneous regression was associated with reversal of T-cell exhaustion features including reduced programmed cell death 1 expression and increased T-cell proliferation. Interestingly, archetypal CLL genomic aberrations including HIST1H1B and TP53 mutations and del(13q14) were found in some spontaneously regressing tumors, but genetic composition remained stable during regression. Conversely, a single case of CLL relapse following spontaneous regression was associated with increased BCR signaling, CLL proliferation, and clonal evolution. These observations indicate that spontaneously regressing CLL appear to undergo a period of proliferation before entering a more quiescent state, and that a complex interaction between genomic alterations and the microenvironment determines disease course. Together, the findings provide novel insight into the biological processes underpinning spontaneous CLL regression, with implications for CLL treatment.

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.

Long-Term Ibrutinib Therapy Reverses CD8+ T Cell Exhaustion in B Cell Chronic Lymphocytic Leukaemia.

Chronic Lymphocytic Leukaemia (CLL) is associated with immune suppression and susceptibility to infection. CD8+ T cell numbers are increased and demonstrate elevated expression of PD-1 and impaired function. The mechanisms driving these features of exhaustion are uncertain but are likely to include chronic immune recognition of tumor and/or infectious agents. We investigated the number, phenotype and function of total and virus-specific CD8+ T cells in 65 patients with CLL and 14 patients undergoing long-term ibrutinib therapy (median 21 months). Ibrutinib substantially reduced the number of both CD3+ T cells and CD8+ T cells. Importantly, this was associated with a reduction in PD-1 expression on CD8+ T cells (median 28 vs. 24%; p = 0.042) and 3.5 fold increase in cytokine production following mitogen stimulation. The influence of ibrutinib on antigen-specific CD8+ T cell function was assessed by HLA-peptide tetramers and revealed increased IFNγ and TNFα cytokine responses following stimulation with CMV or EBV peptides together with a 55% reduction in the frequency of "inflated" virus-specific CD8+ T cells. These findings reveal that long-term ibrutinib therapy is associated with substantial reversal of T cell exhaustion in B-CLL and is likely to contribute to the reduced infection risk seen in association with this agent.