ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.

Histone Deacetylase Inhibitors Directly Modulate T Cell Gene Expression and Signaling and Promote Development of Effector-Exhausted T Cells in Murine Tumors.

Epigenetic regulation plays a crucial role in the development and progression of cancer, including the regulation of antitumor immunity. The reversible nature of epigenetic modifications offers potential therapeutic avenues for cancer treatment. In particular, histone deacetylase (HDAC) inhibitors (HDACis) have been shown to promote antitumor T cell immunity by regulating myeloid cell types, enhancing tumor Ag presentation, and increasing expression of chemokines. HDACis are currently being evaluated to determine whether they can increase the response rate of immune checkpoint inhibitors in cancer patients. Although the potential direct effect of HDACis on T cells likely impacts antitumor immunity, little is known about how HDAC inhibition alters the transcriptomic profile of T cells. In this article, we show that two clinical-stage HDACis profoundly impact gene expression and signaling networks in CD8+ and CD4+ T cells. Specifically, HDACis promoted T cell effector function by enhancing expression of TNF-α and IFN-γ and increasing CD8+ T cell cytotoxicity. Consistently, in a murine tumor model, HDACis led to enrichment of CD8+ T cell subsets with high expression of effector molecules (Prf1, Ifng, Gzmk, and Grmb) but also molecules associated with T cell exhaustion (Tox, Pdcd1, Lag3, and Havcr2). HDACis further generated a tumor microenvironment dominated by myeloid cells with immune suppressive signatures. These results indicate that HDACis directly and favorably augment T cell effector function but also increase their exhaustion signal in the tumor microenvironment, which may add a layer of complexity for achieving clinical benefit in combination with immune checkpoint inhibitors.

TIMEx: tumor-immune microenvironment deconvolution web-portal for bulk transcriptomics using pan-cancer scRNA-seq signatures.

SUMMARY: The heterogeneous cell types of the tumor-immune microenvironment (TIME) play key roles in determining cancer progression, metastasis and response to treatment. We report the development of TIMEx, a novel TIME deconvolution method emphasizing on estimating infiltrating immune cells for bulk transcriptomics using pan-cancer single-cell RNA-seq signatures. We also implemented a comprehensive, user-friendly web-portal for users to evaluate TIMEx and other deconvolution methods with bulk transcriptomic profiles. AVAILABILITY AND IMPLEMENTATION: TIMEx web-portal is freely accessible at http://timex.moffitt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Differential requirement for the IKKß/NF-κB signaling module in regulating TLR- versus RLR-induced type 1 IFN expression in dendritic cells.

Host innate-immune responses are tailored by cell type to control and eradicate specific infectious agents. For example, an acute RNA virus infection can result in high-level expression of type 1 IFNs by both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs), but whereas cDCs preferentially use RIG-I-like receptor (RLR) signaling to produce type 1 IFNs, pDCs predominantly use TLRs to induce these cytokines. We previously found that the IκB kinase ß (IKKß)/NF-κB pathway regulates early IFN-ß expression, but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKKß inhibition and mice deficient in IKKß or canonical NF-κB subunits (p50, RelA/p65, and cRel) to demonstrate that the IKKß/NF-κB axis is critical for virus-induced type 1 IFN expression in pDCs, but not in cDCs. We also reveal a crucial and more general requirement for IKKß/NF-κB in TLR- but not RLR-induced expression of type 1 IFNs and inflammatory cytokines. Together, these findings reveal a previously unappreciated specificity of the IKKß/NF-κB signaling axis in regulation of antimicrobial responses by different classes of pattern recognition receptors, and therefore by individual cell types reliant on particular pattern recognition receptors for their innate-immune transcriptional responses.

Dissection of TBK1 signaling via phosphoproteomics in lung cancer cells.

TANK-binding kinase 1 (TBK1) has emerged as a novel therapeutic target for unspecified subset of lung cancers. TBK1 reportedly mediates prosurvival signaling by activating NF-κB and AKT. However, we observed that TBK1 knockdown also decreased viability of cells expressing constitutively active NF-κB and interferon regulatory factor 3. Basal phospho-AKT level was not reduced after TBK1 knockdown in TBK1-sensitive lung cancer cells, implicating that TBK1 mediates unknown survival mechanisms. To gain better insight into TBK1 survival signaling, we searched for altered phosphoproteins using mass spectrometry following RNAi-mediated TBK1 knockdown. In total, we identified 2,080 phosphoproteins (4,621 peptides), of which 385 proteins (477 peptides) were affected after TBK1 knockdown. A view of the altered network identified a central role of Polo-like kinase 1 (PLK1) and known PLK1 targets. We found that TBK1 directly phosphorylated PLK1 in vitro. TBK1 phosphorylation was induced at mitosis, and loss of TBK1 impaired mitotic phosphorylation of PLK1 in TBK1-sensitive lung cancer cells. Furthermore, lung cancer cell sensitivity to TBK1 was highly correlated with sensitivity to pharmacological PLK inhibition. We additionally found that TBK1 knockdown decreased metadherin phosphorylation at Ser-568. Metadherin was associated with poor outcome in lung cancer, and loss of metadherin caused growth inhibition and apoptosis in TBK1-sensitive lung cancer cells. These results collectively revealed TBK1 as a mitosis regulator through activation of PLK1 and also suggested metadherin as a putative TBK1 downstream effector involved in lung cancer cell survival.

NF-κB is crucial in proximal T-cell signaling for calcium influx and NFAT activation.

In the accepted model of T-cell activation, parallel signal-transduction pathways activate the transcription factors NF-κB, NFAT, and AP-1 to drive clonal expansion of T cells in response to Ag. Genome-wide transcriptional profiling following Ag-induced CD8(+) T-cell activation in C57BL/6 mouse T cells revealed that genes regulated by NFAT were also reduced in the absence of NF-κB p50 and cRel subunits. Importantly, p50(-/-) cRel(-/-) CD8(+) T cells had significantly diminished NFAT and AP-1 activation compared with WT or PKCθ(-/-) CD8(+) T cells. Attenuated NFAT activation after TCR engagement was associated with reduced calcium influx, PLCγ and Zap70 activation. Interestingly, pharmacological bypass of PLCγ-regulated pathways largely rescued p50(-/-) cRel(-/-) T-cell proliferative defects. These results indicate a crucial and unexpected requirement for NF-κB p50 and cRel subunits in proximal TCR signaling and calcium responses. They further suggest that key defects in T cells in the absence of NF-κB pathway components may be due to impaired proximal T-cell signaling.

Interleukin polymorphisms associated with overall survival, disease-free survival, and recurrence in non-small cell lung cancer patients.

Biomarkers based on germline DNA variations could have translational implications by identifying prognostic factors and sub-classifying patients to tailored, patient-specific treatment. To investigate the association between germline variations in interleukin (IL) genes and lung cancer outcomes, we genotyped 251 single nucleotide polymorphisms (SNPs) from 33 different IL genes in 651 non-small cell lung cancer (NSCLC) patients. Analyses were performed to investigate overall survival, disease-free survival, and recurrence. Our analyses revealed 24 different IL SNPs significantly associated with one or more of the lung cancer outcomes of interest. The GG genotype of IL16:rs7170924 was significantly associated with disease-free survival (HR = 0.65; 95% CI 0.50-0.83) and was the only SNP that produced a false discovery rate (FDR) of modest confidence that the association is unlikely to represent a false-positive result (FDR = 0.142). Classification and regression tree (CART) analyses were used to identify potential higher-order interactions. We restricted the CART analyses to the five SNPs that were significantly associated with multiple endpoints (IL1A:rs1800587, IL1B:rs1143634, IL8:s12506479, IL12A:rs662959, and IL13:rs1881457) and IL16:rs7170924 which had the lowest FDR. CART analyses did not yield a tree structure for overall survival; separate CART tree structures were identified for recurrence, based on three SNPs (IL13:rs1881457, IL1B:rs1143634, and IL12A:rs662959), and for disease-free survival, based on two SNPs (IL12A:rs662959 and IL16:rs7170924), which may suggest that these candidate IL SNPs have a specific impact on lung cancer progression and recurrence. These data suggest that germline variations in IL genes are associated with clinical outcomes in NSCLC patients.

Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice.

Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.

Propelling Immunotherapy Combinations Into the Clinic.

Immune checkpoint inhibitors produce durable long-term survival in some patients with advanced melanoma and lung cancer. Better immune targets and combination strategies can harness the immune system by supporting the three elements of a successful T-cell antitumor response: (A) generation of sufficient numbers of antitumor T cells within the lymphoid compartment; (B) effective T-cell trafficking and extravasation out of the lymphoid compartment, through the bloodstream, and into the tumor microenvironment; and (C) T-cell effector function within the tumor microenvironment that is characterized by the ability to bypass immune checkpoints, soluble and metabolic inhibitory factors, and inhibitory cells. Strategies that hold promise include dual immune checkpoint blockade, as well as the combination of immune checkpoint blockade with costimulatory receptor agonists, enhancers of innate immunity, inhibition of indoleamine 2,3-dioxygenase, adoptive T-cell transfer/T-cell engineering, therapeutic vaccines, small-molecule inhibitors, and radiation therapy. Novel, rational clinical trial designs seek to combine targeted agents and one or more immune checkpoint inhibitors, with the goal of producing deep and durable antitumor responses, which thus far have been observed in only a minority of patients.

c-Rel is an essential transcription factor for the development of acute graft-versus-host disease in mice.

Transcription factors of the Rel/NF-κB family are known to play different roles in immunity and inflammation, although the putative role of c-Rel in transplant tolerance and graft-versus-host disease (GVHD) remains elusive. We report here that T cells deficient for c-Rel have a dramatically reduced ability to cause acute GVHD after allogeneic bone marrow transplantation using major and minor histocompatibility mismatched murine models. In the study to understand the underlying mechanisms, we found that c-Rel(-/-) T cells had a reduced ability to expand in lymphoid organs and to infiltrate in GVHD target organs in allogeneic recipients. c-Rel(-/-) T cells were defective in the differentiation into Th1 cells after encountering alloantigens, but were enhanced in the differentiation toward Foxp3(+) regulatory T (Treg) cells. Furthermore, c-Rel(-/-) T cells had largely preserved activity to mediate graft-versus-leukemia response. Taken together, our findings indicate that c-Rel plays an essential role in T cells in the induction of acute GVHD, and suggest that c-Rel can be a potential target for therapeutic intervention in allogeneic hematopoietic cell transplantation in the clinic.

TNFRSF10B polymorphisms and haplotypes associated with increased risk of death in non-small cell lung cancer.

Presently, there are few validated biomarkers that can predict survival or treatment response for non-small cell lung cancer (NSCLC) and most are based on tumor markers. Biomarkers based on germ line DNA variations represent a valuable complementary strategy, which could have translational implications by subclassifying patients to tailored, patient-specific treatment. We analyzed single nucleotide polymorphisms (SNPs) in 53 inflammation-related genes among 651 NSCLC patients. Multivariable Cox proportional hazard models, adjusted for lung cancer prognostic factors, were used to assess the association of genotypes and haplotypes with overall survival. Four of the top 15 SNPs associated with survival were located in the TNF-receptor superfamily member 10b (TNFRSF10B) gene. The T-allele of the top ranked SNP (rs11785599) was associated with a 41% increased risk of death (95% confidence interval [CI] = 1.16-1.70) and the other three TNFRSF10B SNPs (rs1047275, rs4460370 and rs883429) exhibited a 35% (95% CI = 1.11-1.65), 29% (95% CI = 1.06-1.57) and 24% (95% CI = 0.99-1.54) increased risk of death, respectively. Haplotype analyses revealed that the most common risk haplotype (TCTT) was associated with a 78% (95% CI = 1.25-2.54) increased risk of death compared with the low-risk haplotype (CGCC). When the data were stratified by treatment, the risk haplotypes exhibited statistically significantly increased risk of death among patients who had surgery only and no statistically significant effects among patients who had surgery and adjuvant chemotherapy. These data suggest that possessing one or more risk alleles in TNFRSF10B is associated with an increased risk of death. Validated germ line biomarkers may have potential important clinical implications by optimizing patient-specific treatment.

IKKß-induced inflammation impacts the kinetics but not the magnitude of the immune response to a viral vector.

Microbial adjuvants in vaccines activate key transcription factors, including NF-κB and interferon response factors (IRFs). However, the individual role of these transcription factor pathways in promoting adaptive immunity by adjuvants is not clear. It is widely believed that induction of a strong inflammatory response potentiates an adaptive immune response. In this study, we sought to determine whether activation of the pro-inflammatory inhibitor of κB kinase ß (IKKß) canonical NF-κB pathway promoted vaccine-induced immune responses. An adenovirus expressing constitutively activated IKKß (AdIKK) induced robust DC maturation and high expression of key cytokines compared with a control virus. In vivo, AdIKK triggered rapid inflammation after pulmonary infection, increased leukocyte entry into draining LNs, and enhanced early antibody and T-cell responses. Notably, AdIKK did not influence the overall magnitude of the adaptive immune response. These results indicate that induction of inflammation by IKKß/NFκB in this setting impacts the kinetics but not the magnitude of adaptive immune responses. These findings therefore help define the individual role of a key pathway induced by vaccine adjuvants in promoting adaptive immunity.

Combination IFNß and Membrane-Stable CD40L Maximize Tumor Dendritic Cell Activation and Lymph Node Trafficking to Elicit Systemic T-cell Immunity.

Oncolytic virus therapies induce the direct killing of tumor cells and activation of conventional dendritic cells (cDC); however, cDC activation has not been optimized with current therapies. We evaluated the adenoviral delivery of engineered membrane-stable CD40L (MEM40) and IFNß to locally activate cDCs in mouse tumor models. Combined tumor MEM40 and IFNß expression induced the highest cDC activation coupled with increased lymph node migration, increased systemic antitumor CD8+ T-cell responses, and regression of established tumors in a cDC1-dependent manner. MEM40 + IFNß combined with checkpoint inhibitors led to effective control of distant tumors and lung metastases. An oncolytic adenovirus (MEM-288) expressing MEM40 + IFNß  in phase I clinical testing induced cancer cell loss concomitant with enhanced T-cell infiltration and increased systemic presence of tumor T-cell clonotypes in non-small cell lung cancer (NSCLC) patients. This approach to simultaneously target two major DC-activating pathways has the potential to significantly affect the solid tumor immunotherapy landscape.

Distinct roles for the NF-kappa B RelA subunit during antiviral innate immune responses.

Production of type I interferons (IFNs; prominently, IFN-α/ß) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-ß proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnß promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnß expression. Here, we show that RelA instead sustains autocrine IFN-ß signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnß induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnß expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-ß signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process.

NF-kappa B RelA subunit is crucial for early IFN-beta expression and resistance to RNA virus replication.

RNA virus infection results in expression of type 1 IFNs, especially IFN-alpha/beta, which play a crucial role in host antivirus responses. Type 1 IFNs are induced in a cell type-specific manner through TLR and RIG-I-like receptor pathways, both of which activate IFN regulatory factors (IRFs) and NF-kappaB transcription factors. Although NF-kappaB activation and association with the IFN-beta promoter after RNA virus infection is well documented, our previous work showed that, surprisingly, NF-kappaB is not essential for IFN-beta gene expression. Thus, the actual function of NF-kappaB in IFN-beta expression and virus replication is not clear. In this study, we found Newcastle disease virus and vesicular stomatitis virus replication is enhanced in mouse embryonic fibroblasts (MEFs) lacking the NF-kappaB RelA subunit. Increased virus replication was traced to a specific requirement for RelA in early virus-induced IFN-beta expression. At these time points, when IFN-beta expression is ~100-fold less than peak levels, impaired IFN-beta production delayed IFN-induced gene expression, resulting in increased virus replication in RelA(-/-) MEFs. Importantly, our results show that RelA requirement is crucial only when IRF3 activation is low. Thus, high levels of activated IRF3 expression are sufficient for induction of IFN-beta in RelA(-/-) MEFs, transcriptional synergism with the coactivator CREB-binding protein, and rescue of susceptibility to virus. Together, these findings indicate that NF-kappaB RelA is not crucial for regulating overall IFN-beta production, as previously believed; instead, RelA is specifically required only during a key early phase after virus infection, which substantially impacts the host response to virus infection.

Cutting edge: CD28 and c-Rel-dependent pathways initiate regulatory T cell development.

Regulatory T cell (Treg) development proceeds via a two-step process in which naive CD4(+) thymocytes are first converted into CD4(+)CD25(+)CD122(+)GITR(+)Foxp3(-) Treg progenitors, followed by a second step in which IL-2 converts these Treg progenitors into CD4(+)Foxp3(+) Tregs. The costimulatory molecule CD28 is required for efficient Treg development. However, the stage at which CD28 affects Treg development remains undefined. In this article, we demonstrate that Cd28(-/-) mice lack Treg progenitors. Furthermore, the P(187)YAP motif in the cytoplasmic tail of CD28, which links CD28 to Lck activation, is required for this process. In contrast, the Y(170)MNM motif, which links CD28 to PI3K activation, is not required for Treg progenitor development. Finally, the CD28/Lck pathway was shown to activate the NF-kappaB family of transcription factors. We demonstrate that c-Rel, but not NF-kappaB1, promotes the development of Treg progenitors. Thus, a CD28/c-Rel-dependent pathway is involved in initiating Treg development.

Mistic: An open-source multiplexed image t-SNE viewer.

Understanding the complex ecology of a tumor tissue and the spatiotemporal relationships between its cellular and microenvironment components is becoming a key component of translational research, especially in immuno-oncology. The generation and analysis of multiplexed images from patient samples is of paramount importance to facilitate this understanding. Here, we present Mistic, an open-source multiplexed image t-SNE viewer that enables the simultaneous viewing of multiple 2D images rendered using multiple layout options to provide an overall visual preview of the entire dataset. In particular, the positions of the images can be t-SNE or UMAP coordinates. This grouped view of all images allows an exploratory understanding of the specific expression pattern of a given biomarker or collection of biomarkers across all images, helps to identify images expressing a particular phenotype, and can help select images for subsequent downstream analysis. Currently, there is no freely available tool to generate such image t-SNEs.

Phase I Study of Taminadenant (PBF509/NIR178), an Adenosine 2A Receptor Antagonist, with or without Spartalizumab (PDR001), in Patients with Advanced Non-Small Cell Lung Cancer.

PURPOSE: The adenosine 2A receptor (A2AR) mediates the immunosuppressive effects of adenosine in the tumor microenvironment and is highly expressed in non-small cell lung cancer (NSCLC). Taminadenant (PBF509/NIR178) is an A2AR antagonist able to reactivate the antitumor immune response. PATIENTS AND METHODS: In this phase I/Ib, dose-escalation/expansion study, patients with advanced/metastatic NSCLC and ≥1 prior therapy received taminadenant (80-640 mg, orally, twice a day) with or without spartalizumab (anti-programmed cell death-1, 400 mg, i.v., every 4 weeks). Primary endpoints were safety, tolerability, and feasibility of the combination. RESULTS: During dose escalation, 25 patients each received taminadenant alone or with spartalizumab; 19 (76.0%) and 9 (36.0%) had received prior immunotherapy, respectively. Dose-limiting toxicities (all Grade 3) with taminadenant alone were alanine/aspartate aminotransferase increase and nausea [n = 1 (4.0%) each; 640 mg], and in the combination group were pneumonitis [n = 2 (8.0%); 160 and 240 mg] and fatigue and alanine/aspartate aminotransferase increase [n = 1 (4.0%) each; 320 mg]; pneumonitis cases responded to steroids rapidly and successfully. Complete and partial responses were observed in one patient each in the single-agent and combination groups; both were immunotherapy naïve. In the single-agent and combination groups, 7 and 14 patients experienced stable disease; 7 and 6 patients were immunotherapy pretreated, respectively. CONCLUSIONS: Taminadenant, with and without spartalizumab, was well tolerated in patients with advanced NSCLC. The maximum tolerated dose of taminadenant alone was 480 mg twice a day, and 240 mg twice a day plus spartalizumab. Efficacy was neither a primary or secondary endpoint; however, some clinical benefit was noted regardless of prior immunotherapy or programmed cell death ligand-1 status.

Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment.

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.

Utilization of target lesion heterogeneity for treatment efficacy assessment in late stage lung cancer.

RATIONALE: Recent studies have discovered several unique tumor response subgroups outside of response classification by Response Evaluation Criteria for Solid Tumors (RECIST), such as mixed response and oligometastasis. These subtypes have a distinctive property, lesion heterogeneity defined as diversity of tumor growth profiles in RECIST target lesions. Furthermore, many cancer clinical trials have been activated to evaluate various treatment options for heterogeneity-related subgroups (e.g., 29 trials so far listed in clinicaltrials.gov for cancer patients with oligometastasis). Some of the trials have shown survival benefit by tailored treatment strategies. This evidence presents the unmet need to incorporate lesion heterogeneity to improve RECIST response classification. METHOD: An approach for Lesion Heterogeneity Classification (LeHeC) was developed using a contemporary statistical approach to assess target lesion variation, characterize patient treatment response, and translate informative evidence to improving treatment strategy. A mixed effect linear model was used to determine lesion heterogeneity. Further analysis was conducted to classify various types of lesion variation and incorporate with RECIST to enhance response classification. A study cohort of 110 target lesions from 36 lung cancer patients was used for evaluation. RESULTS: Due to small sample size issue, the result was exploratory in nature. By analyzing RECIST target lesion data, the LeHeC approach detected a high prevalence (n = 21; 58%) of lesion heterogeneity. Subgroup classification revealed several informative distinct subsets in a descending order of lesion heterogeneity: mix of progression and regression (n = 7), mix of progression and stability (n = 9), mix of regression and stability (n = 5), and non-heterogeneity (n = 15). Evaluation for association of lesion heterogeneity and RECIST best response classification showed lesion heterogeneity commonly occurred in each response group (stable disease: 16/27; 59%; partial response: 3/5; 60%; progression disease: 2/4; 50%). Survival analysis showed a differential trend of overall survival between heterogeneity and non-heterogeneity in RECIST response groups. CONCLUSION: This is the first study to evaluate lesion heterogeneity, an underappreciated metric, for RECIST application in oncology clinical trials. Results indicated lesion heterogeneity is not an uncommon event. The LeHeC approach could enhance RECIST response classification by utilizing granular lesion level discovery of heterogeneity.