LIGHT (TNFSF14) Costimulation Enhances Myeloid Cell Activation and Antitumor Immunity in the Setting of PD-1/PD-L1 and TIGIT Checkpoint Blockade.

Coinhibition of TIGIT (T cell immunoreceptor with Ig and ITIM domains) and PD-1/PD-L1 (PD-1/L1) may improve response rates compared with monotherapy PD-1/L1 blockade in checkpoint naive non-small cell lung cancer with PD-L1 expression >50%. TIGIT mAbs with an effector-competent Fc can induce myeloid cell activation, and some have demonstrated effector T cell depletion, which carries a clinical liability of unknown significance. TIGIT Ab blockade translates to antitumor activity by enabling PVR signaling through CD226 (DNAM-1), which can be directly inhibited by PD-1. Furthermore, DNAM-1 is downregulated on tumor-infiltrating lymphocytes (TILs) in advanced and checkpoint inhibition-resistant cancers. Therefore, broadening clinical responses from TIGIT blockade into PD-L1low or checkpoint inhibition-resistant tumors, may be induced by immune costimulation that operates independently from PD-1/L1 inhibition. TNFSF14 (LIGHT) was identified through genomic screens, in vitro functional analysis, and immune profiling of TILs as a TNF ligand that could provide broad immune activation. Accordingly, murine and human bifunctional fusion proteins were engineered linking the extracellular domain of TIGIT to the extracellular domain of LIGHT, yielding TIGIT-Fc-LIGHT. TIGIT competitively inhibited binding to all PVR ligands. LIGHT directly activated myeloid cells through interactions with LTßR (lymphotoxin ß receptor), without the requirement for a competent Fc domain to engage Fcγ receptors. LIGHT costimulated CD8+ T and NK cells through HVEM (herpes virus entry mediator A). Importantly, HVEM was more widely expressed than DNAM-1 on T memory stem cells and TILs across a range of tumor types. Taken together, the mechanisms of TIGIT-Fc-LIGHT promoted strong antitumor activity in preclinical tumor models of primary and acquired resistance to PD-1 blockade, suggesting that immune costimulation mediated by LIGHT may broaden the clinical utility of TIGIT blockade.

Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons.

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Topoisomerase 1 inhibition reversibly impairs synaptic function.

Topotecan is a topoisomerase 1 (TOP1) inhibitor that is used to treat various forms of cancer. We recently found that topotecan reduces the expression of multiple long genes, including many neuronal genes linked to synapses and autism. However, whether topotecan alters synaptic protein levels and synapse function is currently unknown. Here we report that in primary cortical neurons, topotecan depleted synaptic proteins that are encoded by extremely long genes, including Neurexin-1, Neuroligin-1, Cntnap2, and GABA(A)ß3. Topotecan also suppressed spontaneous network activity without affecting resting membrane potential, action potential threshold, or neuron health. Topotecan strongly suppressed inhibitory neurotransmission via pre- and postsynaptic mechanisms and reduced excitatory neurotransmission. The effects on synaptic protein levels and inhibitory neurotransmission were fully reversible upon drug washout. Collectively, our findings suggest that TOP1 controls the levels of multiple synaptic proteins and is required for normal excitatory and inhibitory synaptic transmission.

Lipid-Encapsulated mRNAs Encoding Complex Fusion Proteins Potentiate Antitumor Immune Responses.

Lipid nanoparticle (LNP)-encapsulated mRNA has been used for in vivo production of several secreted protein classes, such as IgG, and has enabled the development of personalized vaccines in oncology. Establishing the feasibility of delivering complex multispecific modalities that require higher-order structures important for their function could help expand the use of mRNA/LNP biologic formulations. Here, we evaluated whether in vivo administration of mRNA/LNP formulations of SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT could achieve oligomerization and extend exposure, on-target activity, and antitumor responses comparable with that of the corresponding recombinant fusion proteins. Intravenous infusion of the formulated LNP-encapsulated mRNAs led to rapid and sustained production of functional hexameric proteins in vivo, which increased the overall exposure relative to the recombinant protein controls by ∼28 to 140 fold over 96 hours. High concentrations of the mRNA-encoded proteins were also observed in secondary lymphoid organs and within implanted tumors, with protein concentrations in tumors up to 134-fold greater than with the recombinant protein controls 24 hours after treatment. In addition, SIRPα-Fc-CD40L and TIGIT-Fc-LIGHT mRNAs induced a greater increase in antigen-specific CD8+ T cells in the tumors. These mRNA/LNP formulations were well tolerated and led to a rapid increase in serum and intratumoral IL2, delayed tumor growth, extended survival, and outperformed the activities of benchmark mAb controls. Furthermore, the mRNA/LNPs demonstrated improved efficacy in combination with anti-PD-L1 relative to the recombinant fusion proteins. These data support the delivery of complex oligomeric biologics as mRNA/LNP formulations, where high therapeutic expression and exposure could translate into improved patient outcomes. SIGNIFICANCE: Lipid nanoparticle-encapsulated mRNA can efficiently encode complex fusion proteins encompassing immune checkpoint blockers and costimulators that functionally oligomerize in vivo with extended pharmacokinetics and durable exposure to induce potent antitumor immunity.

Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.

Protein kinase A (PKA)-mediated enhancement of L-type calcium currents (I(Ca,L)) is essential for sympathetic regulation of the heartbeat and is the classic example of channel regulation by phosphorylation, and its loss is a common hallmark of heart failure. Mechanistic understanding of how distinct Ca(V) channel subunits contribute to PKA modulation of I(Ca,L) has been intensely pursued yet remains elusive. Moreover, critical features of this regulation such as its functional reserve (the surplus capacity available for modulation) in the heart are unknown. Here, we use an overexpression paradigm in heart cells to simultaneously identify the impact of auxiliary Ca(V)betas on PKA modulation of I(Ca,L) and to gauge the functional reserve of this regulation in the heart. Ca(V)1.2 channels containing wild-type beta(2a) or a phosphorylation-deficient mutant (beta(2a,AAA)) were equally upregulated by PKA, discounting a necessary role for beta phosphorylation. Nevertheless, channels reconstituted with beta(2a) displayed a significantly diminished PKA response compared with other beta isoforms, an effect explainable by a uniquely higher basal P(o) of beta(2a) channels. Overexpression of all betas increased basal current density, accompanied by a concomitant decrease in the magnitude of PKA regulation. Scatter plots of fold increase in current against basal current density revealed an inverse relationship that was conserved across species and conformed to a model in which a large fraction of channels remained unmodified after PKA activation. These results redefine the role of beta subunits in PKA modulation of Ca(V)1.2 channels and uncover a new design principle of this phenomenon in the heart, vis à vis a limited functional reserve.

A CaVbeta SH3/guanylate kinase domain interaction regulates multiple properties of voltage-gated Ca2+ channels.

Auxiliary Ca(2+) channel beta subunits (Ca(V)beta) regulate cellular Ca(2+) signaling by trafficking pore-forming alpha(1) subunits to the membrane and normalizing channel gating. These effects are mediated through a characteristic src homology 3/guanylate kinase (SH3-GK) structural module, a design feature shared in common with the membrane-associated guanylate kinase (MAGUK) family of scaffold proteins. However, the mechanisms by which the Ca(V)beta SH3-GK module regulates multiple Ca(2+) channel functions are not well understood. Here, using a split-domain approach, we investigated the role of the interrelationship between Ca(V)beta SH3 and GK domains in defining channel properties. The studies build upon a previously identified split-domain pair that displays a trans SH3-GK interaction, and fully reconstitutes Ca(V)beta effects on channel trafficking, activation gating, and increased open probability (P(o)). Here, by varying the precise locations used to separate SH3 and GK domains and monitoring subsequent SH3-GK interactions by fluorescence resonance energy transfer (FRET), we identified a particular split-domain pair that displayed a subtly altered configuration of the trans SH3-GK interaction. Remarkably, this pair discriminated between Ca(V)beta trafficking and gating properties: alpha(1C) targeting to the membrane was fully reconstituted, whereas shifts in activation gating and increased P(o) functions were selectively lost. A more extreme case, in which the trans SH3-GK interaction was selectively ablated, yielded a split-domain pair that could reconstitute neither the trafficking nor gating-modulation functions, even though both moieties could independently engage their respective binding sites on the alpha(1C) (Ca(V)1.2) subunit. The results reveal that Ca(V)beta SH3 and GK domains function codependently to tune Ca(2+) channel trafficking and gating properties, and suggest new paradigms for physiological and therapeutic regulation of Ca(2+) channel activity.

Synapse-specific control of experience-dependent plasticity by presynaptic NMDA receptors.

Sensory experience orchestrates the development of cortical circuitry by adaptively modifying neurotransmission and synaptic connectivity. However, the mechanisms underlying these experience-dependent modifications remain elusive. Here we demonstrate that visual experience suppresses a presynaptic NMDA receptor (preNMDAR)-mediated form of timing-dependent long-term depression (tLTD) at visual cortex layer (L) 4-2/3 synapses. This tLTD can be maintained during development, or reinstated in adulthood, by sensory deprivation. The changes in tLTD are mirrored by changes in glutamate release; visual deprivation enhances both tLTD and glutamate release. These effects require the GluN3A NMDAR subunit, the levels of which are increased by visual deprivation. Further, by coupling the pathway-specific optogenetic induction of tLTD with cell-type-specific NMDAR deletion, we find that visual experience modifies preNMDAR-mediated plasticity specifically at L4-L2/3 synapses.

A single CaVbeta can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels.

Voltage-dependent calcium-channel beta subunits (Ca(V)beta) strongly modulate pore-forming alpha(1) subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P(o)). Despite their central role, it is unclear whether binding of a single Ca(V)beta, or multiple Ca(V)betas, to an alpha(1) subunit governs the two distinct functions. Conventional experiments utilizing coexpression of alpha(1) and Ca(V)beta subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1: 1 stoichiometry by covalently linking Ca(V)beta(2b) to the carboxyl terminus of alpha(1C) (Ca(V)1.2), creating alpha(1C).beta(2b). Recombinant L-type channels reconstituted in HEK 293 cells with alpha(1C).beta(2b) supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed alpha(1C).beta(2b) fully restored channel trafficking to the plasma membrane. Co-transfecting Ca(V)beta(2a) with alpha(1C).beta(2b) had little further impact on function. To rule out the possibility that fused Ca(V)beta(2b) was interacting in trans with neighbouring alpha(1) molecules, alpha(1C).beta(2b) was cotransfected with alpha(1B) (Ca(V)2.2), and pharmacological block with nimodipine showed an absence of alpha(1B) trafficking. These results establish that association of a single Ca(V)beta with a pore-forming alpha(1) subunit captures the functional essence of HVA calcium channels, and introduce alpha(1)-Ca(V)beta fusion proteins as a powerful new tool to probe structure-function mechanisms.

Membrane-associated guanylate kinase-like properties of beta-subunits required for modulation of voltage-dependent Ca2+ channels.

High-voltage-activated Ca2+ channels regulate diverse functions ranging from muscle contraction to synaptic transmission. Association between auxiliary beta- and distinct pore-forming alpha1-subunits is obligatory for forming functional high-voltage-activated Ca2+ channels, yet the structural determinants underlying this interaction remain poorly understood. Recently, homology modeling of Ca(2+)-channel beta1b-subunit identified src homology 3 (SH3) and guanylate kinase (GK) motifs in a tandem arrangement reminiscent of the membrane-associated guanylate kinase (MAGUK) class of scaffolding proteins. However, direct evidence for MAGUK-like properties and their functional implications in beta-subunits is lacking. Here, we show a functional requirement for both SH3 and GK domains in beta2a. Point mutations in either the putative beta2a SH3 or GK domains severely blunted modulation of recombinant L-type channels, showing the importance of both motifs for a functional alpha1-beta interaction. Coexpression of these functionally deficient beta2a-SH3 and GK mutants rescued WT currents, demonstrating trans complementation similar to that observed in MAGUKs. Truncated "hemi-beta2a" subunits, containing either the SH3 or GK domain, were ineffective on their own, but reconstituted WT currents when coexpressed. Moreover, the SH3 and GK domains were found to interact in vitro. These findings reveal MAGUK-like properties in beta-subunits that are critical for alpha1-subunit modulation, revise current models of alpha1-beta association, and predict new physiological dimensions of beta-subunit function.