Hans Christian Reinhardt

Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors.

Treating ATM-deficient cancers with an inhibitor of DNA-PKcs induces apoptosis due to inability to repair double-strand breaks in DNA. Two Wrongs Making a Right for Cancer Treatment When a cell’s DNA is damaged, its normal response is to repair its DNA or undergo apoptosis, a programmed death for cells that are damaged beyond repair. Cancer cells don’t always undergo apoptosis when they should and thus accumulate mutations over time. Even cancer cells, however, need to have some way to repair DNA damage, particularly double-strand breaks, to survive. The two normal mechanisms for such repair are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR requires the function of ATM, a kinase that’s frequently mutated in cancer cells. NHEJ is a more error-prone pathway that does not require ATM but does require another kinase, DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Now, Riabinska et al. show a way to target ATM-mutant cancer by taking advantage of the cells’ need to repair double-strand breaks in DNA. The inhibition of DNA-PKcs in cancers that were already deficient in ATM proved to be very effective for forcing them to undergo apoptosis because they could no longer repair double-strand breaks in DNA at all. DNA-PKcs inhibition did not kill normal cells or cancer cells that had a functioning HR pathway. Thus far, the effects of treating ATM-deficient tumors with DNA-PKcs inhibitors have only been shown in cultured cells and in mice, so this approach still needs to be tested in human patients. This may happen soon because one such inhibitor is already in clinical trials. In the meantime, it looks like making things go wrong in two different DNA repair pathways may yet be the right approach for treating some cancers. When the integrity of the genome is threatened, cells activate a complex, kinase-based signaling network to arrest the cell cycle, initiate DNA repair, or, if the extent of damage is beyond repair capacity, induce apoptotic cell death. The ATM protein lies at the heart of this signaling network, which is collectively referred to as the DNA damage response (DDR). ATM is involved in numerous DDR-regulated cellular responses—cell cycle arrest, DNA repair, and apoptosis. Disabling mutations in the gene encoding ATM occur frequently in various human tumors, including lung cancer and hematological malignancies. We report that ATM deficiency prevents apoptosis in human and murine cancer cells exposed to genotoxic chemotherapy. Using genetic and pharmacological approaches, we demonstrate in vitro and in vivo that ATM-defective cells display strong non-oncogene addiction to DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Further, this dependence of ATM-defective cells on DNA-PKcs offers a window of opportunity for therapeutic intervention: We show that pharmacological or genetic abrogation of DNA-PKcs in ATM-defective cells leads to the accumulation of DNA double-strand breaks and the subsequent CtBP-interacting protein (CtIP)–dependent generation of large single-stranded DNA (ssDNA) repair intermediates. These ssDNA structures trigger proapoptotic signaling through the RPA/ATRIP/ATR/Chk1/p53/Puma axis, ultimately leading to the apoptotic demise of ATM-defective cells exposed to DNA-PKcs inhibitors. Finally, we demonstrate that DNA-PKcs inhibitors are effective as single agents against ATM-defective lymphomas in vivo. Together, our data implicate DNA-PKcs as a drug target for the treatment of ATM-defective malignancies.

Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy

Genetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment.

B-cell-specific conditional expression of Myd88(p.L252P) leads to the development of diffuse large B-cell lymphoma in mice

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40(+) interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625.

Putting the brakes on p53-driven apoptosis

Following genotoxic stress, cells activate a complex, kinase-based signaling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumor suppressor p53 lies at the heart of this DNA damage response. p53 mediates the transactivation of both cell cycle-regulating and pro-apoptotic clusters of target genes. However, it remains incompletely understood which signaling molecules dictate the choice between these two opposing p53-dependent cellular outcomes. Over recent years, numerous regulatory mechanisms impacting on the cellular outcome of p53 signaling have been described. However, no single dominant mechanism has thus far been identified to regulate the cellular choice between p53-driven apoptosis or senescence. The transcriptional regulator AATF has recently emerged as a novel factor impacting on the cellular outcome of the p53 response. Upon genotoxic stress, cytoplasmic pools of MRLC-bound AATF are phosphorylated through the p38MAPK/MK2 checkpoint kinase complex. This AATF phosphorylation results in the disruption of cytoplasmic MRLC3:AATF complexes followed by rapid nuclear localization of AATF. Once in the nucleus, AATF binds to the PUMA, BAX and BAK promoters to repress the DNA damage-induced expression of these pro-apoptotic p53 target genes. Depletion of AATF in tumor cells results in a dramatically enhanced response to DNA-damaging chemotherapeutics, both in vitro and in vivo. Furthermore, focal copy number gains at the AATF locus in neuroblastoma correlate with adverse prognosis and reduced overall survival in this typically p53-proficient malignancy. These data identify the p38/MK2/AATF signaling pathway as a critical repressor of p53-driven apoptosis in tumor cells and implicate this signaling cascade as a novel target for chemotherapy-sensitizing therapeutic efforts.

ATM Deficiency Is Associated with Sensitivity to PARP1- and ATR Inhibitors in Lung Adenocarcinoma

Defects in maintaining genome integrity are a hallmark of cancer. The DNA damage response kinase ATM is frequently mutated in human cancer, but the significance of these events to chemotherapeutic efficacy has not been examined deeply in whole organism models. Here we demonstrate that bi-allelic Atm deletion in mouse models of Kras-mutant lung adenocarcinoma does not affect cisplatin responses. In marked contrast, Atm-deficient tumors displayed an enhanced response to the topoisomerase-II poison etoposide. Moreover, Atm-deficient cells and tumors were sensitive to the PARP inhibitor olaparib. This actionable molecular addiction to functional PARP1 signaling was preserved in models that were proficient or deficient in p53, resembling standard or high-risk genetic constellations, respectively. Atm deficiency also markedly enhanced sensitivity to the ATR inhibitor VE-822. Taken together, our results provide a functional rationale to profile human tumors for disabling ATM mutations, particularly given their impact on PARP1 and ATR inhibitors. Cancer Res; 77(11); 3040-56. ©2017 AACR.

Targeting ATM-deficient CLL through interference with DNA repair pathways.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world and accounts for approximately 30% of adult leukemias and 25% of non-Hodgkin lymphomas. The median age at diagnosis is 72 years. During recent years numerous genetic aberrations have been identified that are associated with an aggressive course of the disease and resistance against genotoxic chemotherapies. The DNA damage-responsive proapoptotic ATM-CHK2-p53 signaling pathway is frequently mutationally inactivated in CLL either through large deletions on chromosome 11q (ATM) or 17p (TP53), or through protein-damaging mutations. Here, we focus on the role of ATM signaling for the immediate DNA damage response, DNA repair and leukemogenesis. We further discuss novel therapeutic concepts for the targeted treatment of ATM-defective CLLs. We specifically highlight the potential use of PARP1 and DNA-PKcs inhibitors for the treatment of ATM-mutant CLL clones. Lastly, we briefly discuss the current state of genetically engineered mouse models of the disease and emphasize the use of these preclinical tools as a common platform for the development and validation of novel therapeutic agents.

ATM Deficiency Generating Genomic Instability Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Therapy-Induced DNA Damage

Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576-90. ©2017 AACR.

Ercc1 Deficiency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Specific Manner.

KRAS-mutant lung adenocarcinoma is among the most common cancer entities and, in advanced stages, typically displays poor prognosis due to acquired resistance against chemotherapy, which is still largely based on cisplatin-containing combination regimens. Mechanisms of cisplatin resistance have been extensively investigated, and ERCC1 has emerged as a key player due to its central role in the repair of cisplatin-induced DNA lesions. However, clinical data have not unequivocally confirmed ERCC1 status as a predictor of the response to cisplatin treatment. Therefore, we employed an autochthonous mouse model of Kras-driven lung adenocarcinoma resembling human lung adenocarcinoma to investigate the role of Ercc1 in the response to cisplatin treatment. Our data show that Ercc1 deficiency in Tp53-deficient murine lung adenocarcinoma induces a more aggressive tumor phenotype that displays enhanced sensitivity to cisplatin treatment. Furthermore, tumors that relapsed after cisplatin treatment in our model develop a robust etoposide sensitivity that is independent of the Ercc1 status and depends solely on previous cisplatin exposure. Our results provide a solid rationale for further investigation of the possibility of preselection of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients might benefit from sequential cisplatin and etoposide chemotherapy. Implications: This study provides a solid rationale for the stratification of lung adenocarcinoma patients according to the functional ERCC1- and mutational TP53 status, where functionally ERCC1-incompetent patients could benefit from sequential cisplatin and etoposide chemotherapy. Mol Cancer Res; 14(11); 1110–23. ©2016 AACR.

AATF suppresses apoptosis, promotes proliferation and is critical for Kras -driven lung cancer

A fundamental principle in malignant tranformation is the ability of cancer cells to escape the naturally occurring cell-intrinsic responses to DNA damage. Tumors progress despite the accumulation of DNA lesions. However, the underlying mechanisms of this tolerance to genotoxic stress are still poorly characterized. Here, we show that replication stress occurs in Kras-driven murine lung adenocarcinomas, as well as in proliferating murine embryonic and adult tissues. We identify the transcriptional regulator AATF/CHE-1 as a key molecule to sustain proliferative tissues and tumor progression in parts by inhibiting p53-driven apoptosis in vivo. In an autochthonous Kras-driven lung adenocarcinoma model, deletion of Aatf delayed lung cancer formation predominantly in a p53-dependent manner. Moreover, targeting Aatf in existing tumors through a dual recombinase strategy caused a halt in tumor progression. Taken together, these data suggest that AATF may serve as a drug target to treat KRAS-driven malignancies.