Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRTs) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and ER stress response via a genetically intact MYC-p19ARF-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide a rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRTs.

Abstract Homozygous deletion of the MTAP gene is one of the most common genetic alterations in cancer, affecting 10-15% of all human cancer. Clinical MTA-cooperative PRMT5 inhibitors, including TNG908 and TNG462, were developed to leverage the well-characterized synthetic lethal interaction between PRMT5 inhibition and MTAP deletion. Indeed, the first disclosed clinical data for an MTA-cooperative PRMT5 inhibitor demonstrated the ability of TNG908 to selectively inhibit PRMT5 in MTAP-deleted tumors while sparing adjacent normal, MTAP-proficient cells in patients. To further explore the mechanism of action of MTA-cooperative PRMT5 inhibitors in MTAP-deleted cells, we employed multiple CRISPR-based editing platforms (CRISPRn, CRISPRi and CRISPRa) to conduct unbiased MTA-cooperative PRMT5 inhibitor anchor screens in vitro and in vivo. A panel of cancer models representing multiple histologies revealed potential PRMT5 inhibitor sensitization and resistance pathways that also include mechanisms that are specific to MTA-cooperative inhibitors. Specifically, the results of these screens identify 1) the effect of MTA/SAM metabolism on MTA-cooperative PRMT5 inhibitors, and 2) identification of potential sub-stratification strategies for patients with MTAP-deleted cancer, and 3) the activity of PRMT5 in anti-apoptotic pathways and synergy between PRMT5 and BCLxL inhibition. Citation Format: Steven Lombardo, Matthew R Tonini, Lauren Grove, Silvia Fenoglio, James Tepper, Binzhang Shen, Hannah Stowe, Shangtao Liu, Samuel R Meier, Ashley H Choi, Tenzing Khendu, Yi Yu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Kimberly J Briggs, Teng Teng. Unbiased in vitro and in vivo drug anchor screens identify mechanisms of resistance and sensitization for MTA-cooperative PRMT5 inhibitors in MTAP-deleted cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B098.

Supplementary Table 4. STR profiles for cell lines used in this study.

Cholinergic interneurons (CINs) are essential elements of striatal circuits and functions. Although acetylcholine signaling via muscarinic receptors (mAChRs) has been well studied, more recent data indicate that postsynaptic nicotinic receptors (nAChRs) located on striatal GABAergic interneurons (GINs) are equally critical. One example is that CIN stimulation induces large disynaptic inhibition of striatal projection neurons (SPNs) mediated by nAChR activation of GINs. Although these circuits are ideally positioned to modulate striatal output, the neurons involved are not definitively identified because of an incomplete mapping of CINs-GINs interconnections. Here, we show that CINs modulate four GINs populations via an intricate mechanism involving co-activation of presynaptic and postsynaptic mAChRs and nAChRs. Using optogenetics, we demonstrate the participation of tyrosine hydroxylase-expressing GINs in the disynaptic inhibition of SPNs via heterotypic electrical coupling with neurogliaform interneurons. Altogether, our results highlight the importance of CINs in regulating GINs microcircuits via complex synaptic/heterosynaptic mechanisms.

The functions of cortical networks are progressively established during development by series of events shaping the neuronal connectivity. Synaptic elimination, which consists of removing the supernumerary connections generated during the earlier stages of cortical development, is one of the latest stages in neuronal network maturation. The semaphorin 3F coreceptors neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) may play an important role in the functional maturation of the cerebral cortex by regulating the excess dendritic spines on cortical excitatory neurons. Yet, the identity of the connections eliminated under the control of Nrp2/PlxnA3 signaling is debated, and the importance of this synaptic refinement for cortical functions remains poorly understood. Here, we show that Nrp2/PlxnA3 controls the spine densities in layer 4 (L4) and on the apical dendrite of L5 neurons of the sensory and motor cortices. Using a combination of neuroanatomical, ex vivo electrophysiology, and in vivo functional imaging techniques in Nrp2 and PlxnA3 KO mice of both sexes, we disprove the hypothesis that Nrp2/PlxnA3 signaling is required to maintain the ectopic thalamocortical connections observed during embryonic development. We also show that the absence of Nrp2/PlxnA3 signaling leads to the hyperexcitability and excessive synchronization of the neuronal activity in L5 and L4 neuronal networks, suggesting that this system could participate in the refinement of the recurrent corticocortical connectivity in those layers. Altogether, our results argue for a role of semaphorin–Nrp2/PlxnA3 signaling in the proper maturation and functional connectivity of the cerebral cortex, likely by controlling the refinement of recurrent corticocortical connections. SIGNIFICANCE STATEMENT The function of a neuronal circuit is mainly determined by the connections that neurons establish with one another during development. Understanding the mechanisms underlying the establishment of the functional connectivity is fundamental to comprehend how network functions are implemented, and to design treatments aiming at restoring damaged neuronal circuits. Here, we show that the cell surface receptors for the family of semaphorin guidance cues neuropilin 2 (Nrp2) and plexin-A3 (PlxnA3) play an important role in shaping the functional connectivity of the cerebral cortex likely by trimming the recurrent connections in layers 4 and 5. By removing the supernumerary inputs generated during early development, Nrp2/PlxnA3 signaling reduces the neuronal excitability and participates in the maturation of the cortical network functions.

The striatum mediates a broad range of cognitive and motor functions. Within the striatum, recently discovered tyrosine hydroxylase expressing interneurons (THINs) provide a source of intrastriatal synaptic connectivity that is critical for regulating striatal activity, yet the role of THIN's in behavior remains unknown. Given the important role of the striatum in reward-based behaviors, we investigated whether loss of striatal THINs would impact instrumental behavior in mice. We selectively ablated striatal THINs in TH-Cre mice using chemogenetic techniques, and then tested THIN-lesioned or control mice on three reward-based striatal-dependent instrumental tests: (a) progressive ratio test; (b) choice test following selective-satiety induced outcome devaluation; (c) outcome reinstatement test. Both striatal-THIN-lesioned and control mice acquired an instrumental response for flavored food pellets, and their behavior did not differ in the progressive ratio test, suggesting intact effort to obtain rewards. However, striatal THIN lesions markedly impaired choice performance following selective-satiety induced outcome devaluation. Unlike control mice, THIN-lesioned mice did not adjust their choice of actions following a change in outcome value. In the outcome reinstatement test THIN-lesioned and control mice showed response invigoration by outcome presentation, suggesting the incentive properties of outcomes were not disrupted by THIN lesions. Overall, we found that striatal THIN lesions selectively impaired goal-directed behavior, while preserving motoric and appetitive behaviors. These findings are the first to describe a function of striatal THINs in reward-based behavior, and further illustrate the important role for intrastriatal interneuronal connectivity in behavioral functions ascribed to the striatum more generally.

The main excitatory inputs to the striatum arising from the cortex and the thalamus innervate both striatal spiny projection neurons and interneurons. These glutamatergic inputs to striatal GABAergic interneurons have been suggested to regulate the spike timing of striatal projection neurons via feedforward inhibition. Understanding how different excitatory inputs are integrated within the striatal circuitry and how they regulate striatal output is crucial for understanding basal ganglia function and related behaviors. Here, using VGLUT2 mice from both sexes, we report the existence of a glutamatergic projection from the mesencephalic locomotor region to the striatum that avoids the spiny neurons and selectively innervates interneurons. Specifically, optogenetic activation of glutamatergic axons from the pedunculopontine nucleus induced monosynaptic excitation in most recorded striatal cholinergic interneurons and GABAergic fast-spiking interneurons. Optogenetic stimulation in awake head-fixed mice consistently induced an increase in the firing rate of putative cholinergic interneurons and fast-spiking interneurons. In contrast, this stimulation did not induce excitatory responses in spiny neurons but rather disynaptic inhibitory responses ex vivo and a decrease in their firing rate in vivo, suggesting a feedforward mechanism mediating the inhibition of spiny projection neurons through the selective activation of striatal interneurons. Furthermore, unilateral stimulation of pedunculopontine nucleus glutamatergic axons in the striatum induced ipsilateral head rotations consistent with the inhibition of striatal output neurons. Our results demonstrate the existence of a unique interneuron-specific midbrain glutamatergic input to the striatum that exclusively recruits feedforward inhibition mechanisms.SIGNIFICANCE STATEMENT Glutamatergic inputs to the striatum have been shown to target both striatal projection neurons and interneurons and have been proposed to regulate spike timing of the projection neurons in part through feedforward inhibition. Here, we reveal the existence of a midbrain source of glutamatergic innervation to the striatum, originating in the pedunculopontine nucleus. Remarkably, this novel input selectively targets striatal interneurons, avoiding the projection neurons. Furthermore, we show that this selective innervation of interneurons can regulate the firing of the spiny projection neurons and inhibit the striatal output via feedforward inhibition. Together, our results describe a unique source of excitatory innervation to the striatum which selectively recruits feedforward inhibition of spiny neurons without any accompanying excitation.

The classical view of striatal GABAergic interneuron function has been that they operate as largely independent, parallel, feedforward inhibitory elements providing inhibitory inputs to spiny projection neurons (SPNs). Much recent evidence has shown that the extrinsic innervation of striatal interneurons is not indiscriminate but rather very specific, and that striatal interneurons are themselves interconnected in a cell type-specific manner. This suggests that the ultimate effect of extrinsic inputs on striatal neuronal activity depends critically on synaptic interactions within interneuronal circuitry. Here, we compared the cortical and thalamic input to two recently described subtypes of striatal GABAergic interneurons, tyrosine hydroxylase-expressing interneurons (THINs), and spontaneously active bursty interneurons (SABIs) using transgenic TH-Cre and Htr3a-Cre mice of both sexes. Our results show that both THINs and SABIs receive strong excitatory input from the motor cortex and the thalamic parafascicular nucleus. Cortical optogenetic stimulation also evokes disynaptic inhibitory GABAergic responses in THINs but not in SABIs. In contrast, optogenetic stimulation of the parafascicular nucleus induces disynaptic inhibitory responses in both interneuron populations. However, the short-term plasticity of these disynaptic inhibitory responses is different suggesting the involvement of different intrastriatal microcircuits. Altogether, our results point to highly specific interneuronal circuits that are selectively engaged by different excitatory inputs.

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

The striatum constitutes the main input structure of the basal ganglia and receives two major excitatory glutamatergic inputs, from the cortex and the thalamus. Excitatory cortico- and thalamostriatal connections innervate the principal neurons of the striatum, the spiny projection neurons (SPNs), which constitute the main cellular input as well as the only output of the striatum. In addition, corticostriatal and thalamostriatal inputs also innervate striatal interneurons. Some of these inputs have been very well studied, for example the thalamic innervation of cholinergic interneurons and the cortical innervation of striatal fast-spiking interneurons, but inputs to most other GABAergic interneurons remain largely unstudied, due in part to the relatively recent identification and characterization of many of these interneurons. In this review, we will discuss and reconcile some older as well as more recent data on the extrinsic excitatory inputs to striatal interneurons. We propose that the traditional feed-forward inhibitory model of the cortical input to the fast-spiking interneuron then inhibiting the SPN, often assumed to be the prototype of the main functional organization of striatal interneurons, is incomplete. We provide evidence that the extrinsic innervation of striatal interneurons is not uniform but shows great cell-type specificity. In addition, we will review data showing that striatal interneurons are themselves interconnected in a highly cell-type-specific manner. These data suggest that the impact of the extrinsic inputs on striatal activity critically depends on synaptic interactions within interneuronal circuitry.

The recent availability of different transgenic mouse lines coupled with other modern molecular techniques has led to the discovery of an unexpectedly large cellular diversity and synaptic specificity in striatal interneuronal circuitry. Prior research has described three spontaneously active interneuron types in mouse striatal slices: the cholinergic interneuron, the neuropeptide Y-low threshold spike interneuron, and the tyrosine hydroxylase interneurons (THINs). Using transgenic Htr3a-Cre mice, we now characterize a fourth population of spontaneously active striatal GABAergic interneurons termed spontaneously active bursty interneurons (SABIs) because of their unique burst-firing pattern in cell-attached recordings. Although they bear some qualitative similarity in intrinsic electrophysiological properties to THINs in whole-cell recordings, detailed analysis revealed significant differences in many intrinsic properties and in their morphology. Furthermore, all previously identified striatal GABAergic interneurons have been shown to innervate striatal spiny projection neurons (SPNs), contributing to the suggestion that the principal function of striatal GABAergic interneurons is to provide feedforward inhibition to SPNs. Here, very surprisingly, paired recordings show that SABIs do not innervate SPNs significantly. Further, optogenetic inhibition of striatal Htr3a-Cre interneurons triggers barrages of IPSCs in SPNs. We hypothesize that these IPSCs result from disinhibition of a population of GABAergic interneurons with activity that is constitutively suppressed by the SABIs. We suggest that the SABIs represent the first example of a striatal interneuron-selective interneuron and, further, that their existence, along with previously defined interneuronal networks, may participate in the formation of SPN ensembles observed by others. SIGNIFICANCE STATEMENT Before ∼2010, the main function of the three known subtypes of striatal GABAergic interneurons was assumed to mediate feedforward inhibition of the spiny neurons (SPNs). During the past decade, we and others have described several novel populations of striatal GABAergic interneurons and their synaptic connections and have shown that striatal interneurons and SPNs interact through extensive and highly cell-type-specific connections that form specialized networks. Here, we describe a novel population of striatal GABAergic interneuron and provide several lines of evidence suggesting that it represents the first interneuron-selective interneuron in striatum. Striatal interneurons and their synaptic connections are suggested to play an important role in the formation of ensembles of striatal SPNs interconnected by inhibitory axon collaterals.

The striatum is the main input nucleus of the basal ganglia and is a key site of sensorimotor integration. While the striatum receives extensive excitatory afferents from the cerebral cortex, the influence of different cortical areas on striatal circuitry and behavior is unknown. Here, we find that corticostriatal inputs from whisker-related primary somatosensory (S1) and motor (M1) cortex differentially innervate projection neurons and interneurons in the dorsal striatum and exert opposing effects on sensory-guided behavior. Optogenetic stimulation of S1-corticostriatal afferents in ex vivo recordings produced larger postsynaptic potentials in striatal parvalbumin (PV)-expressing interneurons than D1- or D2-expressing spiny projection neurons (SPNs), an effect not observed for M1-corticostriatal afferents. Critically, in vivo optogenetic stimulation of S1-corticostriatal afferents produced task-specific behavioral inhibition, which was bidirectionally modulated by striatal PV interneurons. Optogenetic stimulation of M1 afferents produced the opposite behavioral effect. Thus, our results suggest opposing roles for sensory and motor cortex in behavioral choice via distinct influences on striatal circuitry.

Recent discoveries of striatal GABAergic interneurons require a new conceptualization of the organization of intrastriatal circuitry and their cortical and thalamic inputs. We investigated thalamic inputs to the two populations of striatal neuropeptide Y (NPY) interneurons, plateau low threshold spike (PLTS) and NPY-neurogliaform (NGF) cells. Optogenetic activation of parafascicular inputs evokes suprathreshold monosynaptic glutamatergic excitation in NGF interneurons and a disynaptic, nicotinic excitation through cholinergic interneurons. In contrast, the predominant response of PLTS interneurons is a disynaptic inhibition dependent on thalamic activation of striatal tyrosine hydroxylase interneurons (THINs). In contrast, THINs do not innervate NGF or fast spiking interneurons, showing significant specificity in THINs outputs. Chemospecific ablation of THINs impairs prepulse inhibition of the acoustic startle response suggesting an important behavioural role of this disynaptic pathway. Our findings demonstrate that the impact of the parafascicular nucleus on striatal activity and some related behaviour critically depend on synaptic interactions within interneuronal circuits.

Depletion of Smarcb1 activates the Myc network of signalling cascades, increasing protein metabolism and activation of survival pathways allowing highly aggressive Kras-independent pancreatic cancer cells to develop. In pancreatic cancer models driven by Kras mutations, the authors find that a subpopulation of cancer cells can become independent of Kras signalling and instead acquire a mesenchymal phenotype driven by a Smarcb1–Myc network. This change is associated with an increase in protein metabolism, inhibition of which renders tumours sensitive to inhibition of this metabolic adaptation. These findings suggest a potential strategy for treating pancreatic cancer. Malignant neoplasms evolve in response to changes in oncogenic signalling1. Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance2,3. Here we determine the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1–Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Increased protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α–MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

Current treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1-4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.

The nigrostriatal dopamine (DA) system is crucial to a large array of behaviors ranging from voluntary motor function to higher cognitive processes. Nigrostriatal neurons in vivo fire in three distinct patterns, modulated by afferent input. At least 70% of the inputs to nigral DA neurons are GABAergic, arising from the striatum, the globus pallidus, the rostromedial tegmental nucleus, and axon collaterals from the substantia nigra pars reticulata (SNr) projection neurons. Manipulation of these inputs, in particular those originating in SNr, produces dramatic effects on nigrostriatal neuron firing patterns. Since many or most of the afferents to the substantia nigra synapse on both nigrostriatal DA neurons and SNr GABAergic projection neurons, the response of nigral DA neurons to both excitatory and inhibitory afferents reflects a complex interplay between the intrinsic properties of the nigrostriatal neurons, the patterns and rate of the afferent input, and the synaptic interactions between SNr GABAergic neurons and the nigrostriatal DA neurons.

Dopamine neurons in the ventral tegmental area (VTA) receive cholinergic innervation from brainstem structures associated with either movement or reward. While cholinergic neurons of the pedunculopontine nucleus (PPN) carry an associative/motor signal, those of the laterodorsal tegmental nucleus (LDT) convey limbic information. Here we used optogenetic methods combined with in vivo juxtacellular recording and labeling to dissect the influence of brainstem cholinergic innervation of distinct subpopulations of neurons in the VTA. We found that LDT cholinergic axons selectively enhance the bursting activity of mesolimbic dopamine neurons that are excited by aversive stimulation. In contrast, PPN cholinergic axons activate and change the discharge properties of VTA neurons that are integrated in distinct functional circuits and are inhibited by aversive stimulation. While both structures conveyed a reinforcing signal, they had opposite roles in locomotion. Our results demonstrate that two modes of cholinergic transmission operate in the VTA and segregate neurons involved in different reward circuits.