Current immunotherapy paradigms aim to reinvigorate CD8
T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck ...squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV
), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV
HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV
HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies.
Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don't. A new data integrative approach, ...based on graphical models, was applied on our multi-modal -omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells (especially in ovarian cancer for which olaparib is FDA-approved), but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors.
BackgroundCurrent FDA-approved immunotherapies aim to reinvigorate CD8+ T cells, but the contribution of the humoral arm of the immune response in human cancer remains poorly understood. B cells ...within tissues can mediate anti-tumor immunity and regulate immune responses by presenting antigen and producing tumor-specific antibodies and immunomodulatory cytokines. Head and neck squamous cell carcinoma (HNSCC) can be induced by human papillomavirus (HPV+) and carcinogens such as tobacco and alcohol (HPV-), and the immune infiltrate is quite distinct in the two etiologies, in particular, increased B cells in HPV+ HNSCC patients. Further, increased B cells in HNSCC patients correlate with improved patient survival. Our study seeks to differentiate B cell phenotype, function and location in HPV+ and HPV- HNSCC to identify putative B cell-centric immunotherapeutic targets.MethodsWe utilized a multi-level approach to clearly categorize B cells in HNSCC patients. Single cell RNA sequencing (scRNAseq) was performed on CD45+ tumor infiltrating lymphocytes (TIL) from HPV+ and HPV- HNSCC patients. HNSCC TIL and PBL were stained via spectral cytometry (Cytek Aurora,25 parameters) for unbiased analysis of B cell subsets via computational spectral unmixing. Paraffin embedded slides from HNSCC primary tumors were utilized for multispectral immunofluorescence (mIF) to identify tertiary lymphoid structures (TLS) and identify differences in HPV+ and HPV- disease.ResultsWe demonstrated distinct trajectories for B cells in HPV+ and HPV- disease. HPV- HNSCC tumors mainly contained memory B cells and plasma cells, while the B cells in HPV+ HNSCC were naïve and germinal center (GC). Further, we quantified B cells and CD4+ T cells in TLS, and germinal center-like TLS were associated with improved outcome in HPV+ disease. We also observed that transcriptional and protein expression of Semaphorin A (SEMA4a) was restricted to GC B cells and increased on GC B cells in HNSCC patients compared to healthy tonsils. Additionally, we identified distinct waves of gene expression in GC B cells in HNSCC tumors, ultimately revealing a novel transitional state for GC B cells in the tumor microenvironment (TME).ConclusionsUnderstanding B cell function in human cancers and how different TMEs influence B cells and TLS are important for devising novel therapeutic options for cancer patients. Ultimately, development of therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.
The t(5;17) variant of acute promeylocytic leukemia (APL) expresses a fusion of nucleophosmin (NPM) with the retinoic acid receptor alpha (RARA). We have previously shown that NPM-RAR is a binding ...partner of the tumor necrosis factor (TNF) receptor type-I-associated DEATH domain protein, TRADD. Binding of TNF to its receptor, TNF-R, induces recruitment of TRADD, and subsequent recruitment of a cascade of proteins that ultimate activate caspase 3, nuclear factor κB (NFκB) and c-Jun N-terminal kinase (JNK). We have previously shown that NPM-RAR interaction with TRADD blocks TNF activation of caspase 3, caspase 8, poly(ADP-ribose) polymerase (PARP) cleavage and, ultimately, apoptosis. We now report that NPM-RAR expression is permissive for TNF activation of NFκB and JNK. We propose that inhibition of TNF activation of apoptosis, while preserving TNF activation of NFκB and JNK pathways that stimulate cell growth and survival, represents a novel mechanism through which NPM-RAR contributes to development of the leukemic phenotype.
Despite the success of immune checkpoint blockade therapy, few strategies sufficiently overcome immunosuppression within the tumor microenvironment (TME). Targeting regulatory T cells (T
) is ...challenging, because perturbing intratumoral T
function must be specific enough to avoid systemic inflammatory side effects. Thus, no T
-targeted agents have proven both safe and efficacious in patients with cancer. Neuropilin-1 (NRP1) is recognized for its role in supporting intratumoral T
function while being dispensable for peripheral homeostasis. Nonetheless, little is known about the biology of human NRP1
T
and the signals that regulate NRP1 expression. Here, we report that NRP1 is preferentially expressed on intratumoral T
across six distinct cancer types compared to healthy donor peripheral blood peripheral blood lymphocyte (PBL) and site-matched, noncancer tissue. Furthermore, NRP1
T
prevalence is associated with reduced progression-free survival in head and neck cancer. Human NRP1
T
have broad activation programs and elevated suppressive function. Unlike mouse T
, we demonstrate that NRP1 identifies a transient activation state of human T
driven by continuous T cell receptor (TCR) signaling through the mitogen-activated protein kinase pathway and interleukin-2 exposure. The prevalence of NRP1
T
in patient PBL correlates with the intratumoral abundance of NRP1
T
and may indicate higher disease burden. These findings support further clinical evaluation of NRP1 as a suitable therapeutic target to enhance antitumor immunity by inhibiting T
function in the TME.
Abstract
B cells can regulate immune responses by presenting antigen, producing antigen-specific antibodies and immunomodulatory cytokines. Their role in the anti-tumor immune response is poorly ...understood. However, in many cancers including head and neck squamous cell carcinoma (HNSCC), intratumoral B cells correlate with better survival. HNSCC has two distinct etiologies (HPV−) and (HPV+) where patients who are HPV+ have increased B cell infiltration and respond better to therapy. We hypothesized that (1) intratumoral B cell phenotype is different between HPV+ and HPV− HNSCC (2) location within the tumor microenvironment (TME) is distinct and (3) antibodies produced by intratumoral B cell in HPV+ HNSCC are specific for viral antigens.
Using single-cell RNA sequencing and spectral flow cytometry, we observed that B cell signatures in HPV− HNSCC patients were predominantly memory B cells and plasma cells, while the signatures in HPV+ HNSCC were naïve and germinal center (GC) B cells. Further, we quantified B cells in tertiary lymphoid structures (TLS) using multispectral immunofluorescence, and the presence of GC-rich TLS were increased in HPV+ patients. In fact, GC-rich TLS within the tumor of HPV+ patients correlated with increased overall survival. Overall, high enrichment for GC B cells were positively associated with longer progression-free survival. Antibodies produced by intratumoral B cells from HPV+ patients were positive for HPV viral antigens. Ultimately, characterization of B cell phenotype and function in HNSCC is important for devising new therapeutic options for cancer patients. Specifically, therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.
Abstract 5117
Acute Promyelocytic Leukemia (APL) is characterized by recurrent translocations of 17q21, which result in expression of fusion proteins of the retinoic acid receptor alpha (RARα). We ...have been studying the t(5;17)(q35;q21) variant of APL, which fuses the N-terminal 117 amino acids of nucleophosmin (NPM) to the C-terminus of RARα. NPM-RAR is capable of binding to retinoic acid response elements (RAREs), and we have previously shown that NPM-RAR can recruit both corepressor and coactivator complexes to retinoic acid target promoters, to modulate transcription in a ligand-dependent fashion, though with a different sensitivity to ligand than RARα. The N-terminal NPM-sequence of NPM-RAR contains a dimerization domain capable of mediating not only homodimerization of the fusion protein, but also heterodimerization with wild-type NPM, to potentially modulate NPM functionality. NPM serves as a molecular chaperone, and binds to a diverse set of proteins. Amongst these are the p50 and p65 subunits of NFκB. NPM has been shown to serve as a coactivator for the NFκB transcriptional complex. We hypothesized that NPM-RAR might interact with NPM to modulate NFκB dependent transcription. We focused on NFκB regulation of the Superoxide Dismutase 2 (MnSOD, SOD2) promoter, since NPM regulation of NFκB mediated transcription has been most extensively studied in this model system. We found that NPM-RAR enhances both basal and TNFα-induced transcription of an NFκB-reporter construct (containing a minimal MnSOD promoter/enhancer) in HepG2 and Hela cells. This effect was not dependent upon translocation of NFκB to the nucleus, indicating that the positive effect on NFκB was mediated downstream of the canonical activation pathway of NFκB. The MnSOD promoter does not contain a consensus RARE, and this effect was not seen in cells transfected with RARα, suggesting that this effect is not mediated through the DNA binding motifs of the NPM-RAR C-terminal RARα sequences. NPM-RAR does not directly interact with NFκB; preliminary experiments using siRNA to down-regulate NPM indicate that NPM-RAR enhancement of MnSOD transcription is dependent upon the presence of wild-type NPM. This leads us to propose a model whereby NPM-RAR binds to the NPM/NFκB transcriptional complex through interaction with NPM, to enhance NFκB mediated transcription.
No relevant conflicts of interest to declare.
The variant acute promyelocytic leukemia (APL) translocation t(5;17)(q35;q21) fuses the N-terminus of nucleophosmin (NPM1) to the retinoic acid receptor alpha (RARA). We found that ectopic NPM1-RARA ...expression decreased TP53 protein levels in target cells. NPM1-RARA impaired TP53-dependent transcription. Cells expressing NPM1-RARA were more resistant to apoptotic stimuli. This work identifies the TP53 tumor suppressor as a novel target through which NPM1-RARA impacts leukemogenesis, and confirms the importance of impairment of TP53 in establishment of the APL phenotype.
Abstract The t(5;17)(q35;q21) APL variant results in expression of a fusion protein linking the N-terminus of nucleophosmin (NPM) to the C-terminus of the retinoic acid receptor alpha (RAR). We have ...previously shown that NPM-RAR is capable of binding to DNA either as a homodimer or heterodimer with RXR. To determine the biological significance of NPM-RAR/RXR interaction, we developed two mutants of NPM-RAR that showed markedly diminished ability to bind RXR. U937 subclones expressing the NPM-RAR mutants showed significantly less inhibition of vitamin D3/TGFbeta-induced differentiation, compared with NPM-RAR. These results support the hypothesis that RXR interaction is necessary for NPM-RAR-mediated myeloid maturation arrest.
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