p90RSK pathway inhibition synergizes with cisplatin in TMEM16A overexpressing head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.

Polyamine transport inhibition and cisplatin synergistically enhance tumor control through oxidative stress in murine head and neck cancer models.

Background: Surgery and/or platinum-based chemoradiation remain standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While these therapies are effective in a subset of patients, a substantial proportion experience recurrence or treatment resistance. As cisplatin mediates cytotoxicity through oxidative stress while polyamines play a role in redox regulation, we posited that combining cisplatin with polyamine transport inhibitor, AMXT-1501, would increase oxidative stress and tumor cell death in HNSCC cells. Methods: Cell proliferation was measured in syngeneic mouse HNSCC cell lines treated with cisplatin ± AMXT-1501. Synergy was determined by administering cisplatin and AMXT-1501 at a ratio of 1:10 to cancer cells in vitro . Cancer cells were transferred onto mouse flanks to test the efficacy of treatments in vivo . Reactive oxygen species (ROS) were measured. Cellular apoptosis was measured with flow cytometry using Annexin V/PI staining. High-performance liquid chromatography (HPLC) was used to quantify polyamines in cell lines. Cell viability and ROS were measured in the presence of exogenous cationic amino acids. Results: The combination of cisplatin and AMXT-1501 synergize in vitro on HNSCC cell lines. In vivo combination treatment resulted in tumor growth inhibition greater than either treatment individually. The combination treatment increased ROS production and induced apoptotic cell death. HPLC revealed the synergistic mechanism was independent of intracellular polyamine levels. Supplementation of cationic amino acids partially rescued cancer cell viability and reduced ROS. Conclusion: AMXT-1501 enhances the cytotoxic effects of cisplatin in vitro and in vivo in aggressive HNSCC cell lines through a polyamine-independent mechanism.

Lysosomal mitochondrial interaction promotes tumor growth in squamous cell carcinoma of the head and neck.

Tumor growth and proliferation are regulated by numerous mechanisms. Communication between intracellular organelles has recently been shown to regulate cellular proliferation and fitness. The way lysosomes and mitochondria communicate with each other (lysosomal/mitochondrial interaction) is emerging as a major determinant of tumor proliferation and growth. About 30% of squamous carcinomas (including squamous cell carcinoma of the head and neck, SCCHN) overexpress TMEM16A, a calcium-activated chloride channel, which promotes cellular growth and negatively correlates with patient survival. TMEM16A has recently been shown to drive lysosomal biogenesis, but its impact on mitochondrial function is unclear. Here, we show that (1) patients with high TMEM16A SCCHN display increased mitochondrial content specifically complex I; (2) In vitro and in vivo models uniquely depend on mitochondrial complex I activity for growth and survival; (3) ß-catenin/NRF2 signaling is a critical linchpin that drives mitochondrial biogenesis, and (4) mitochondrial complex I and lysosomal function are codependent for proliferation. Taken together, our data demonstrate that LMI drives tumor proliferation and facilitates a functional interaction between lysosomes and mitochondria. Therefore, inhibition of LMI may serve as a therapeutic strategy for patients with SCCHN.

Next generation self-replicating RNA vectors for vaccines and immunotherapies.

RNA technology has recently come to the forefront of innovative medicines and is being explored for a wide range of therapies, including prophylactic and therapeutic vaccines, biotherapeutic protein expression and gene therapy. In addition to conventional mRNA platforms now approved for prophylactic SARS-CoV2 vaccines, synthetic self-replicating RNA vaccines are currently being evaluated in the clinic for infectious disease and oncology. The prototypical srRNA vectors in clinical development are derived from alphaviruses, specifically Venezuelan Equine Encephalitis Virus (VEEV). While non-VEEV alphaviral strains have been explored as single cycle viral particles, their use as synthetic vectors largely remains under-utilized in clinical applications. Here we describe the potential commonalities and differences in synthetic alphaviral srRNA vectors in host cell interactions, immunogenicity, cellular delivery, and cargo expression. Thus, unlike the current thinking that VEEV-based srRNA is a one-size-fits-all platform, we argue that a new drug development approach leveraging panels of customizable, synthetic srRNA vectors will be required for clinical success.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

CD8+ T cells mediate protection against Zika virus induced by an NS3-based vaccine.

Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.

Cationic HDL mimetics enhance in vivo delivery of self-replicating mRNA.

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development.

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

A forward genetic screen reveals roles for Nfkbid, Zeb1, and Ruvbl2 in humoral immunity.

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble, cellophane, and Worker ascribed to mutations in Nfkbid, Zeb1, and Ruvbl2, respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid, is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.

Divergent transcriptional programming of class-specific B cell memory by T-bet and RORα.

Antibody class defines function in B cell immunity, but how class is propagated into B cell memory remains poorly understood. Here we demonstrate that memory B cell subsets unexpectedly diverged across antibody class through differences in the effects of major transcriptional regulators. Conditional genetic deletion of the gene encoding the transcription factor T-bet selectively blocked the formation and antigen-specific response of memory B cells expressing immunoglobulin G2a (IgG2a) in vivo. Cell-intrinsic expression of T-bet regulated expression of the transcription factor STAT1, steady-state cell survival and transcription of IgG2a-containing B cell antigen receptors (BCRs). In contrast, the transcription factor RORα and not T-bet was expressed in IgA(+) memory B cells, with evidence that knockdown of RORα mRNA expression and chemical inhibition of transcriptional activity also resulted in lower survival and BCR expression of IgA(+) memory B cells. Thus, divergent transcriptional regulators dynamically maintain subset integrity to promote specialized immune function in class-specific memory B cells.

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme.

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%-8%. Interestingly, ZFNickases targeting the AAVS1 "safe harbor" locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition-suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

Molecular programming of B cell memory.

The development of high-affinity B cell memory is regulated through three separable phases, each involving antigen recognition by specific B cells and cognate T helper cells. Initially, antigen-primed B cells require cognate T cell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, B cells with variant B cell receptors must access antigens and present them to germinal centre T helper cells to enter long-lived memory B cell compartments. Following antigen recall, memory B cells require T cell help to proliferate and differentiate into plasma cells. A recent surge of information - resulting from dynamic B cell imaging in vivo and the elucidation of T follicular helper cell programmes - has reshaped the conceptual landscape surrounding the generation of memory B cells. In this Review, we integrate this new information about each phase of antigen-specific B cell development to describe the newly unravelled molecular dynamics of memory B cell programming.

Derivation and characterization of a simian immunodeficiency virus SIVmac239 variant with tropism for CXCR4.

Like human immunodeficiency virus type 1 (HIV-1), most simian immunodeficiency virus (SIV) strains use CCR5 to establish infection. However, while HIV-1 can acquire the ability to use CXCR4, SIVs that utilize CXCR4 have rarely been reported. To explore possible barriers against SIV coreceptor switching, we derived an R5X4 variant, termed 239-ST1, from the R5 clone SIVmac239 by serially passaging virus in CD4(+) CXCR4(+) CCR5(-) SupT1 cells. A 239-ST1 env clone, designated 239-ST1.2-32, used CXCR4 and CCR5 in cell-cell fusion and reporter virus infection assays and conferred the ability for rapid, cytopathic infection of SupT1 cells to SIVmac239. Viral replication was inhibitable by the CXCR4-specific antagonist AMD3100, and replication was abrogated in a novel CXCR4(-) SupT1 line. Surprisingly, parental SIVmac239 exhibited low-level replication in SupT1 cells that was not observed in CXCR4(-) SupT1 cells. Only two mutations in the 239-ST1.2-32 Env, K47E in the C1 domain and L328W in the V3 loop, were required for CXCR4 use in cell-cell fusion assays, although two other V3 changes, N316K and I324M, improved CXCR4 use in infection assays. An Env cytoplasmic tail truncation, acquired during propagation of 239-ST1 in SupT1 cells, was not required. Compared with SIVmac239, 239-ST1.2-32 was more sensitive to neutralization by five of seven serum and plasma samples from SIVmac239-infected rhesus macaques and was approximately 50-fold more sensitive to soluble CD4. Thus, SIVmac239 can acquire the ability to use CXCR4 with high efficiency, but the changes required for this phenotype may be distinct from those for HIV-1 CXCR4 use. This finding, along with the increased neutralization sensitivity of this CXCR4-using SIV, suggests a mechanism that could select strongly against this phenotype in vivo.

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases.

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

An improved zinc-finger nuclease architecture for highly specific genome editing.

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.