Search

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC–attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations.Significance:NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

Supplementary Figure S1. This supplementary figure shows that SNF5 KD in normal human fibroblasts does not alter mRNA levels of SWI/SNF components.

Table shows genes with significantly altered expression by RNA-seq after BRG1 or BRM knockout.

MIB/MS data

Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP–Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs.Implications: SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation. Mol Cancer Res; 12(11); 1574–85. ©2014 AACR.

Supplementary Figure 1 shows BRG1 deficiency activates transcription of a subset of NRF2 target genes in second H358 BRG1 knockdown cell line; Supplementary Figure 2 shows western blot of BRG1 and BRM knockout samples used for RNA-seq.

Supplemental Figure Legends

Abemeciclib competitive MIB/MS TMT dose response

Supplemental Table 5. SCCOHT-1 ABPP data; Supplemental Table 6. BIN67 ABPP data.

Genes differentially expressed in LUSC TCGA in High CDR1-Low miR-671-5p tumors compared to Low CDR1-High miR-671 tumors.

The relationship between NFE2L2 mutation and radiation resistance in OC cancer. Kaplan-Meier analysis of PFS and OSin OC patients treated with surgery and postoperative (chemo)radiotherapy.

CB-839 overcomes the Nrf2E79Q-induced radiation resistance through regulating TLR4-mediated cytokine changes.

Mutational landscape and details of NFE2L2 gene mutation in a cohort of oral cavity cancer patients treated with surgery and postoperative (chemo)radiotherapy at Stanford University.

Nrf2 mutation leads to extrinsic radiation resistance through recruitment of PMN-MDSC in tumors.

IC50 summary of validated drug hits in SCCOHT cell lines.

Supplemental Table 1 shows patients distribution in OC cohorts from Stanford University. Supplemental Table 2 shows primers for mutagenesis. Supplemental Table 3 shows primers for real-time PCR. Supplemental Table 4 shows gene mutations identified by targeted next generation sequencing in a cohort of oral cavity cancer patients treated with surgery and postoperative (chemo)radiotherapy at Stanford University. Supplemental Table 5 shows tumor growth delay of indicated MOC1 xenografts using 1000mm3 tumor size as a proxy.

Time lapse imaging of RUSH assay in SK-MES-1-CDR1 cells transfected with Ii-Str_ssSBP-EGFP (green) and Golgi marker mApple-SiT (magenta) beginning 5 min after biotin addition.

Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT.Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%.Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Supplementary Methods, Figures, and Tables

Time lapse imaging of RUSH assay in SK-MES-1-EV cells transfected with Ii-Str_ssSBP-EGFP (green) and Golgi marker mApple-SiT (magenta) beginning 5 min after biotin addition.

Supplemental Figure 1. COV434-pIND20-Brg1 SMARCA4 induction optimization; Supplemental Figure 2. Kinase hits from siRNA screen mapped to kinome dendrogram; Supplemental Figure 3. ClueGo analysis of validated siRNA screen hits.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC–attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations.Significance:NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

CB-839 overcomes RT resistance in Nrf2E79Q MOC1 cells through regulating the tumor microenvironment.

Somatic mutation of NFE2L2 leads to Nrf2 activation and promotes intrinsic radiation resistance in human head and neck squamous cell carcinoma (HNSCC).

Supplemental Materials and Methods

Lung squamous carcinoma (LUSC) is a highly metastatic disease with a poor prognosis. Using an integrated screening approach, we found that miR-671-5p reduces LUSC metastasis by inhibiting a circular RNA (circRNA), CDR1as. Although the putative function of circRNA is through miRNA sponging, we found that miR-671-5p more potently silenced an axis of CDR1as and its antisense transcript, cerebellar degeneration related protein 1 (CDR1). Silencing of CDR1as or CDR1 significantly inhibited LUSC metastases and CDR1 was sufficient to promote migration and metastases. CDR1, which directly interacted with adaptor protein 1 (AP1) complex subunits and coatomer protein I (COPI) proteins, no longer promoted migration upon blockade of Golgi trafficking. Therapeutic inhibition of the CDR1as/CDR1 axis with miR-671-5p mimics reduced metastasis in vivo. This report demonstrates a novel role for CDR1 in promoting metastasis and Golgi trafficking. These findings reveal an miRNA/circRNA axis that regulates LUSC metastases through a previously unstudied protein, CDR1.Significance:This study shows that circRNA, CDR1as, promotes lung squamous migration, metastasis, and Golgi trafficking through its complimentary transcript, CDR1.

NFE2L2 mutation leads to extrinsic radiation resistance.

XLSX file - 20K, Database of KEAP1 mutations in cancer.

Supplementary Figure 2. This figure shows additional characterization of global nucleosomal positioning changes in Brg1 KD cell lines.

PDF file - 603K, SQCC KEAP1 mutant characterization.

Supplementary Table 2. Differentially expressed genes were identified between BRG1 mutant and BRG1 wild-type NSCLC cell lines, as described in Materials and Methods. Using an FDR threshold of .025, a total of 135 genes were down-regulated in the BRG1 mutant cell lines.

XLSX file - 11K, Mutagenesis primers and TCGA tumor codes.

XLSX file - 891K, Filtered and unfiltered mass spectrometry data.

PDF file - 72K

PDF file - 587K, KEAP1 mutants differentially bind to protein interactors.

Supplementary Figure 3. This figure shows additional characterization of nucleosome positioning positions changes around transcription start sites in Brg1 KD cell lines.

NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in NRF2 localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in KEAP1 occur commonly in human cancer, where KEAP1 may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 KEAP1 mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these “super-binder” mutants exhibited reduced degradation of NRF2. Cell-based and in vitro biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q “superbinder” mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between KEAP1 and NRF2 in cancer and provide new insight into KEAP1 mechanics. Cancer Res; 74(3); 808–17. ©2013 AACR.

Supplementary Figure 1. This figure shows validation of H358 and H441 Brg1 knockdown cell lines.

Supplementary Table and Figure Legends

Supplementary Figure 4. The figure shows kernal-smoothed data for nucleosome positioning about transcription start sites in the Brg1 KD human NSCLC cell line and in mouse SNF5 and Brg1 KO cells.

Supplementary Table 1. Differentially expressed genes were identified, as described in Material and Methods. Genes altered by BRG1 KD- Genes whose expression either increased or decreased in the H358 Brg1i.1 and H358 Brgi.2 cell lines; Upregulated and unregulated genes- We identified 316 genes that showed increased expression in both H358 Brg1i.1 and H358 Brg1i.2 cell lines. We also identified 300 genes that did not show altered expression in the Brg1i.2 cell line.

Supplementary Figure 5. This figure shows nucleosome positioning around 2 genes downregulated after Brg1 KD.

Homodimers are the most abundant type of enzyme in cells and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on the similar event at the second site, the most informative state is the asymmetric single bound (lig(1)) form, yet it tends to be elusive thermodynamically. Here we obtain milligram quantities of lig(1) of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu((I)-)catalyzed azide–alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins, and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig(1) intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but should also facilitate other structural applications where specific linkage of proteins is helpful.