Lou, Zhenkun

STK38, a reader of histone H4 ufmylation, regulates ATM activation.

Abstract Purpose: NOTCH signaling is associated with tumorigenesis, mutagenesis, and immune tolerance in non–small cell lung cancer (NSCLC), indicating its association with the clinical benefit of immune checkpoint inhibitors (ICI). We hypothesized that NOTCH mutation in NSCLC might be a robust predictor of immunotherapeutic efficacy. Experimental Design: Multiple-dimensional data including genomic, transcriptomic, and clinical data from cohorts of NSCLC internal and public cohorts involving immunotherapeutic patients were analyzed. Polymorphism Phenotyping v2 (PolyPhen-2) system was performed to determine deleterious NOTCH mutation (del-NOTCHmut). Further investigation on molecular mechanism was performed in The Cancer Genome Atlas (TCGA) data via CIBERSORT and gene set enrichment analysis. Results: Our 3DMed cohort (n = 58) and other four cohorts (Rizvi, POPLAR/OAK, Van Allen, and MSKCC; n = 1,499) uncovered marked correlation between NOTCH1/2/3 mutation and better ICI outcomes in EGFR/ALKWT population, including objective response rate (2.20-fold, P = 0.001), progression-free survival [HR, 0.61; 95% confidence interval (CI), 0.46–0.81; P = 0.001], and overall survival (HR, 0.56; 95% CI, 0.32–0.96; P = 0.035). Del-NOTCHmut exhibited better predictive function than non-deleterious NOTCH mutation, potentially via greater transcription of genes related to DNA damage response and immune activation. Del-NOTCHmut was not linked with prognosis in TCGA cohorts and chemotherapeutic response, but was independently associated with immunotherapeutic benefit, delineating the predictive, but not prognostic, utility of del-NOTCHmut. Conclusions: This work distinguishes del-NOTCHmut as a potential predictor to favorable ICI response in NSCLC, highlighting the importance of genomic profiling in immunotherapy. More importantly, our results unravel a possibility of personalized combination immunotherapy as adding NOTCH inhibitor to ICI regimen in NSCLC, for the optimization of ICI treatment in clinical practice.

We clarify that DNA damage triggers autophagy by CHK2-FOXK axis, and misregulation of this pathway leads to chemoresistance.

Abstract Abstract 691 Malignant plasma cells in multiple myeloma (MM) patients display a variety of recurrent genetic abnormalities. In this regard, tumor cells in approximately 15% of all MM patients will exhibit a translocation involving the immunoglobulin (Ig) heavy chain locus at 14q32 and the short arm of chromosome 4. The breakpoint on chromosome 4 (4p16) frequently results in overexpression of FGFR3 and/or full-length or truncated versions of the multiple myeloma SET domain protein, MMSET. MM patients with t(4;14) translocations are considered to have high-risk disease. MMSET has been shown to have histone methyltransferase activity and we have recently shown that this protein plays a pivotal role in DNA repair and maintenance of genetic stability (1). Thus, dysregulation of MMSET may result in aberrant responses to DNA damage, which may be related to the poor prognosis of MM patients with t(4;14) translocations. The MMSET gene is also known as the Wolf-Hirschhorn syndrome candidate (WHSC1) gene. Expression of the WHSC1 gene is uniformly misregulated due to haploinsufficiency in patients with Wolf-Hirschhorn syndrome (WHS) resulting in characteristic facial features and developmental disorders. Of great interest, WHS patients also display significant antibody deficiencies and IgG and IgA deficiencies are particularly frequent. Currently, the underlying cause of antibody deficiencies in WHS patients remains unknown. However, our recent studies have shown that a robust DNA repair process in germinal center B cells is required for fertile antibody maturation processes (2). This observation, taken together with our recent discover that MMSET regulates the recruitment of 53BP1 to sites of DNA damage through its histone methyltransferase activity during DNA damage repair (1), suggested to us the hypothesis that MMSET may also be critically involved in Ig gene maturation, particularly as it concerns class switch recombination, a process known to result from double strand DNA breaks and subsequent effective DNA repair. By using shRNA knockdown technology in murine lymphoma cell line CH12F3 cells, which can be specifically induced to switch from IgM to IgA expression ex vivo by CD40 ligand stimulation in the presence of IL-4 and TGFb, we clearly demonstrate that downregulation of MMSET expression by shRNA significantly impaired class switch recombination from IgM to IgA. While it plays no detectable roles in cell viability, proliferation, or apoptosis, we found that MMSET is important for histone methylation at H3K36 and H4K20 sites of the Igh loci, which in turn modulate the recruitment of 53BP1 to the Igh loci as well as the transcription of the Igh switch regions, leading to defective class switch recombination. Further DNA sequence analysis of post-switched Sm-Sa junctions from MMSET compromised cells showed a significant increase in microhomology suggesting that homologous recombination (HR) repair is alternatively used as a compensatory mechanism during DNA repair of the switch region when MMSET is absent. Our results suggest that defective CSR caused by MMSET deficiency may underpin the antibody deficiency phenotype in WHS patients. Furthermore, our results showing that MMSET expression dose dictates the usage choice between two competing DNA repair pathways, i.e., error-prone non-homologous end joining (NHEJ) and error-free HR, may also suggest that MMSET overexpression in MM may favor usage of the NHEJ pathway therefore leading to more error-prone DNA repair and possibly additional genetic damage. Disclosures: No relevant conflicts of interest to declare.

Abstract Triple-negative breast cancer (TNBC) is characterized by elevated locoregional recurrence risk despite aggressive local therapies. New tumor-specific radiosensitizers are needed. We hypothesized that the ATR inhibitor, VX-970 (now known as M6620), would preferentially radiosensitize TNBC. Noncancerous breast epithelial and TNBC cell lines were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, patient-derived xenograft (PDX) models generated prospectively as part of a neoadjuvant chemotherapy study from either baseline tumor biopsies or surgical specimens with chemoresistant residual disease were assessed for sensitivity to fractionated radiotherapy, VX-970, or the combination. To explore potential response biomarkers, exome sequencing was assessed for germline and/or somatic alterations in homologous recombination (HR) genes and other alterations associated with ATR inhibitor sensitivity. VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the radiotherapy-induced G2–M checkpoint, delayed resolution of DNA double-strand breaks, and reduced colony formation after radiotherapy in TNBC cells relative to normal-like breast epithelial cells. In vivo, VX-970 did not exhibit significant single-agent activity at the dose administered even in the context of genomic alterations predictive of ATR inhibitor responsiveness, but significantly sensitized TNBC PDXs to radiotherapy. Exome sequencing and functional testing demonstrated that combination therapy was effective in both HR-proficient and -deficient models. PDXs established from patients with chemoresistant TNBC were also highly radiosensitized. In conclusion, VX-970 is a tumor-specific radiosensitizer for TNBC. Patients with residual TNBC after neoadjuvant chemotherapy, a subset at particularly high risk of relapse, may be ideally suited for this treatment intensification strategy. Mol Cancer Ther; 17(11); 2462–72. ©2018 AACR.

Abstract Purpose: TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. Patients with residual TNBC after neoadjuvant chemotherapy (NAC) have a high risk of locoregional recurrence despite aggressive subsequent local therapies, including RT. Thus, new radiosensitizers that address chemoresistant TNBC (i.e residual disease after NAC) are needed. ATR plays a key role in the cellular response to replication stress and DNA damage. TNBC is characterized by G1 checkpoint loss, homologous recombination deficiency (HRD), and other features which increase dependence on ATR pathway signaling. We hypothesized that VX-970, a selective inhibitor of ATR, would effectively radiosensitize TNBC. Methods: Cell lines representing varying subtypes of TNBC (MDA-MB-231, HCC1806, and BT-549) and the normal breast epithelial cell line MCF10a were investigated in clonogenic survival, cell cycle, and DNA damage signaling and repair assays. In addition, Mayo Clinic (MC)TNBC1 and MCTNBC2, patient derived xenograft models (PDXs) generated prospectively from baseline and chemoresistant residual disease surgical specimens after NAC, respectively, of unique patients in the Breast Cancer Genome Guided Therapy Study were analyzed. Tumors were injected into hind-legs of athymic nude mice and animals were randomized to: 1) Vehicle 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX-970 was administered daily 1 hour prior to RT at a dose of 60 mg/kg. Exome sequencing was assessed for germ-line and/or somatic alterations in HR genes and an ex-vivo RAD51 foci formation assay was applied to confirm the functional status of HR in each PDX model. Results: In vitro, VX-970 preferentially inhibited ATR-Chk1-CDC25a signaling, abrogated the RT-induced G2/M checkpoint, delayed resolution of γH2AX and 53BP1 foci and reduced colony formation after RT in MDA-MB-231, HCC1806, and BT-549 relative to MCF10a. In vivo, VX-970 did not exhibit single agent activity at the dose administered but markedly sensitized both MCTNBC1 and MCTNBC2 to RT, suggesting that VX-970 could overcome chemoresistant TNBC biology. For MCTNBC1 and MCTNBC2, the median time to tumor tripling was 26 vs 49 days (p=0.002) and 25 vs 43 days (p=0.006) for the RT and RT + VX-970 groups, respectively. Ex-vivo RAD51 foci formation was robust in MCTNBC1, a PDX without HR gene alterations. In contrast, there was no RT-induced RAD51 foci formation in MCTNBC2, suggesting a BRCA1 mutation in the corresponding human tumor resulted in loss of HR function in that model. Conclusion: VX-970 + RT effectively radiosensitized TNBC PDXs established from both pre-treatment biopsy specimens and chemoresistant residual tumor samples and in both HR deficient and HR proficient settings. ATR inhibition should be investigated in the clinic in combination with RT for patients with residual TNBC after NAC. Citation Format: Robert W. Mutter, Mohamed M. Kahila, Xinyi Tu, Qin Zhou, Jia Yu, Krishna R. Kalari, Liewei Wang, Judy C. Boughey, Matthew P. Goetz, Jann N. Sarkaria, Zhenkun Lou. ATR inhibition is a promising radiosensitizing strategy for chemotherapy-resistant triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 840.

Significance ES cells possess the unique capacity to self-renew as well as differentiate into specialized cell types. It is known that transcription factors and chromatin regulators regulate the cell-fate choices during differentiation. We report unexpectedly that Asf1a, a histone chaperone involved in nucleosome assembly, regulates mouse ES cell differentiation. Mechanistically, we show that Asf1a functions in nucleosome disassembly to resolve the bivalent chromatin domains at lineage-specific genes for gene activation during differentiation. These insights will likely be applicable for understanding human ES cell differentiation and regenerative medicine.

ABSTRACT In somatic cells, H2afx and Mdc1 are close functional partners in DNA repair and damage response. However, it is not known whether they are also involved in the maintenance of genome integrity in meiosis. By analyzing chromosome dynamics in H2afx−/− spermatocytes, we found that the synapsis of autosomes and X-Y chromosomes was impaired in a fraction of cells. Such defects correlated with an abnormal recombination profile. Conversely, Mdc1 was dispensable for the synapsis of the autosomes and played only a minor role in X-Y synapsis, compared with the action of H2afx. This suggested that those genes have non-overlapping functions in chromosome synapsis. However, we observed that both genes play a similar role in the assembly of MLH3 onto chromosomes, a key step in crossover formation. Moreover, we show that H2afx and Mdc1 cooperate in promoting the activation of the recombination-dependent checkpoint, a mechanism that restrains the differentiation of cells with unrepaired DSBs. This occurs by a mechanism that involves P53. Overall, our data show that, in male germ cells, H2afx and Mdc1 promote the maintenance of genome integrity. This article has an associated First Person interview with the first author of the paper.