First-in-human dose escalation trial to evaluate the clinical safety and efficacy of an anti-MAGEA1 autologous TCR-transgenic T cell therapy in relapsed and refractory solid tumors.

RATIONALE OF THE TRIAL: Although the use of engineered T cells in cancer immunotherapy has greatly advanced the treatment of hematological malignancies, reaching meaningful clinical responses in the treatment of solid tumors is still challenging. We investigated the safety and tolerability of IMA202 in a first-in-human, dose escalation basket trial in human leucocyte antigen A*02:01 positive patients with melanoma-associated antigen A1 (MAGEA1)-positive advanced solid tumors. TRIAL DESIGN: The 2+2 trial design was an algorithmic design based on a maximally acceptable dose-limiting toxicity (DLT) rate of 25% and the sample size was driven by the algorithmic design with a maximum of 16 patients. IMA202 consists of autologous genetically modified cytotoxic CD8+ T cells expressing a T cell receptor (TCR), which is specific for a nine amino acid peptide derived from MAGEA1. Eligible patients underwent leukapheresis, T cells were isolated, transduced with lentiviral vector carrying MAGEA1-specific TCR and following lymphodepletion (fludarabine/cyclophosphamide), infused with a median of 1.4×109 specific T cells (range, 0.086×109-2.57×109) followed by interleukin 2. SAFETY OF IMA202: No DLT was observed. The most common grade 3-4 adverse events were cytopenias, that is, neutropenia (81.3%), lymphopenia (75.0%), anemia (50.0%), thrombocytopenia (50.0%) and leukopenia (25.0%). 13 patients experienced cytokine release syndrome, including one grade 3 event. Immune effector cell-associated neurotoxicity syndrome was observed in two patients and was grade 1 in both. EFFICACY OF IMA202: Of the 16 patients dosed, 11 (68.8%) patients had stable disease (SD) as their best overall response (Response Evaluation Criteria in Solid Tumors V.1.1). Five patients had initial tumor shrinkage in target lesions and one patient with SD experienced continued shrinkage in target lesions for 3 months in total but had to be classified as progressive disease due to progressive non-target lesions. IMA202 T cells were persistent in peripheral blood for several weeks to months and were also detectable in tumor tissue. Peak persistence was higher in patients who received higher doses. CONCLUSION: In conclusion, IMA202 had a manageable safety profile, and it was associated with biological and potential clinical activity of MAGEA1-targeting genetically engineered TCR-T cells in a poor prognosis, multi-indication solid tumor cohort. TRIAL REGISTRATION NUMBERS: NCT04639245, NCT05430555.

Inhibition of Karyopherin ß1-Mediated Nuclear Import Disrupts Oncogenic Lineage-Defining Transcription Factor Activity in Small Cell Lung Cancer.

Genomic studies support the classification of small cell lung cancer (SCLC) into subtypes based on the expression of lineage-defining transcription factors ASCL1 and NEUROD1, which together are expressed in ∼86% of SCLC. ASCL1 and NEUROD1 activate SCLC oncogene expression, drive distinct transcriptional programs, and maintain the in vitro growth and oncogenic properties of ASCL1 or NEUROD1-expressing SCLC. ASCL1 is also required for tumor formation in SCLC mouse models. A strategy to inhibit the activity of these oncogenic drivers may therefore provide both a targeted therapy for the predominant SCLC subtypes and a tool to investigate the underlying lineage plasticity of established SCLC tumors. However, there are no known agents that inhibit ASCL1 or NEUROD1 function. In this study, we identify a novel strategy to pharmacologically target ASCL1 and NEUROD1 activity in SCLC by exploiting the nuclear localization required for the function of these transcription factors. Karyopherin ß1 (KPNB1) was identified as a nuclear import receptor for both ASCL1 and NEUROD1 in SCLC, and inhibition of KPNB1 led to impaired ASCL1 and NEUROD1 nuclear accumulation and transcriptional activity. Pharmacologic targeting of KPNB1 preferentially disrupted the growth of ASCL1+ and NEUROD1+ SCLC cells in vitro and suppressed ASCL1+ tumor growth in vivo, an effect mediated by a combination of impaired ASCL1 downstream target expression, cell-cycle activity, and proteostasis. These findings broaden the support for targeting nuclear transport as an anticancer therapeutic strategy and have implications for targeting lineage-transcription factors in tumors beyond SCLC. SIGNIFICANCE: The identification of KPNB1 as a nuclear import receptor for lineage-defining transcription factors in SCLC reveals a viable therapeutic strategy for cancer treatment.

ASCL1, NKX2-1, and PROX1 co-regulate subtype-specific genes in small-cell lung cancer.

Lineage-defining transcription factors (LTFs) play key roles in small-cell lung cancer (SCLC) pathophysiology. Delineating the LTF-regulated genes operative in SCLC could provide a road map to identify SCLC dependencies. We integrated chromatin landscape and transcriptome analyses of patient-derived SCLC preclinical models to identify super-enhancers (SEs) and their associated genes in the ASCL1-, NEUROD1-, and POU2F3-high SCLC subtypes. We find SE signatures predict LTF-based classification of SCLC, and the SE-associated genes are enriched with those defined as common essential genes in DepMap. In addition, in ASCL1-high SCLC, we show ASCL1 complexes with NKX2-1 and PROX1 to co-regulate genes functioning in NOTCH signaling, catecholamine biosynthesis, and cell-cycle processes. Depletion of ASCL1 demonstrates it is a key dependency factor in preclinical SCLC models and directly regulates multiple DepMap-defined essential genes. We provide LTF/SE-based subtype-specific gene sets for SCLC for further therapeutic investigation.

ASCL1 represses a SOX9+ neural crest stem-like state in small cell lung cancer.

ASCL1 is a neuroendocrine lineage-specific oncogenic driver of small cell lung cancer (SCLC), highly expressed in a significant fraction of tumors. However, ∼25% of human SCLC are ASCL1-low and associated with low neuroendocrine fate and high MYC expression. Using genetically engineered mouse models (GEMMs), we show that alterations in Rb1/Trp53/Myc in the mouse lung induce an ASCL1+ state of SCLC in multiple cells of origin. Genetic depletion of ASCL1 in MYC-driven SCLC dramatically inhibits tumor initiation and progression to the NEUROD1+ subtype of SCLC. Surprisingly, ASCL1 loss promotes a SOX9+ mesenchymal/neural crest stem-like state and the emergence of osteosarcoma and chondroid tumors, whose propensity is impacted by cell of origin. ASCL1 is critical for expression of key lineage-related transcription factors NKX2-1, FOXA2, and INSM1 and represses genes involved in the Hippo/Wnt/Notch developmental pathways in vivo. Importantly, ASCL1 represses a SOX9/RUNX1/RUNX2 program in vivo and SOX9 expression in human SCLC cells, suggesting a conserved function for ASCL1. Together, in a MYC-driven SCLC model, ASCL1 promotes neuroendocrine fate and represses the emergence of a SOX9+ nonendodermal stem-like fate that resembles neural crest.

Cell-autonomous immune gene expression is repressed in pulmonary neuroendocrine cells and small cell lung cancer.

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed "variant" due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings may help guide the design of treatment regimens in SCLC.

Phosphoprotein-based biomarkers as predictors for cancer therapy.

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. nonsensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

Exposure to mild blast forces induces neuropathological effects, neurophysiological deficits and biochemical changes.

Direct or indirect exposure to an explosion can induce traumatic brain injury (TBI) of various severity levels. Primary TBI from blast exposure is commonly characterized by internal injuries, such as vascular damage, neuronal injury, and contusion, without external injuries. Current animal models of blast-induced TBI (bTBI) have helped to understand the deleterious effects of moderate to severe blast forces. However, the neurological effects of mild blast forces remain poorly characterized. Here, we investigated the effects caused by mild blast forces combining neuropathological, histological, biochemical and neurophysiological analysis. For this purpose, we employed a rodent blast TBI model with blast forces below the level that causes macroscopic neuropathological changes. We found that mild blast forces induced neuroinflammation in cerebral cortex, striatum and hippocampus. Moreover, mild blast triggered microvascular damage and axonal injury. Furthermore, mild blast caused deficits in hippocampal short-term plasticity and synaptic excitability, but no impairments in long-term potentiation. Finally, mild blast exposure induced proteolytic cleavage of spectrin and the cyclin-dependent kinase 5 activator, p35 in hippocampus. Together, these findings show that mild blast forces can cause aberrant neurological changes that critically impact neuronal functions. These results are consistent with the idea that mild blast forces may induce subclinical pathophysiological changes that may contribute to neurological and psychiatric disorders.

Different Originating Cells Underlie Intertumoral Heterogeneity in Lung Neuroendocrine Tumors.

Studies in genetically engineered mouse models of neuroendocrine lung cancer suggest that differences in cells of origin underlie subtype variations in this class of cancers. These findings highlight the concept that the same driver mutations introduced into different cells of origin lead to tumors with the same histology but dramatically different metastatic programs and potentially different therapeutic responses. Cancer Discov; 8(10); 1216-8. ©2018 AACR See related article by Yang et al., p. 1316.

Identifying a missing lineage driver in a subset of lung neuroendocrine tumors.

Tumor heterogeneity of a primary histologic cancer type has major implications for cancer research and therapeutics. An important and understudied aspect of this heterogeneity is the role of transcription factors that serve as "lineage oncogenes" in a tumor type. A demonstration that different subgroups have distinct dependencies on lineage-specific transcription factors is highlighted in a relatively homogenous cancer type: the pulmonary neuroendocrine cancer small cell lung carcinoma (SCLC). Identification of these factors is providing new insights into the origin of the heterogeneity and subtype-specific vulnerabilities in SCLC and provides a template for studying heterogeneity in other cancer types.

Intrinsic DNA binding properties demonstrated for lineage-specifying basic helix-loop-helix transcription factors.

During development, transcription factors select distinct gene programs, providing the necessary regulatory complexity for temporal and tissue-specific gene expression. How related factors retain specificity, especially when they recognize the same DNA motifs, is not understood. We address this paradox using basic helix-loop-helix (bHLH) transcription factors ASCL1, ASCL2, and MYOD1, crucial mediators of lineage specification. In vivo, these factors recognize the same DNA motifs, yet bind largely different genomic sites and regulate distinct transcriptional programs. This suggests that their ability to identify regulatory targets is defined either by the cellular environment of the partially defined lineages in which they are endogenously expressed, or by intrinsic properties of the factors themselves. To distinguish between these mechanisms, we directly compared the chromatin binding properties of this subset of bHLH factors when ectopically expressed in embryonic stem cells, presenting them with a common chromatin landscape and cellular components. We find that these factors retain distinct binding sites; thus, specificity of binding is an intrinsic property not requiring a restricted landscape or lineage-specific cofactors. Although the ASCL factors and MYOD1 have some distinct DNA motif preference, it is not sufficient to explain the extent of the differential binding. All three factors can bind inaccessible chromatin and induce changes in chromatin accessibility and H3K27ac. A reiterated pattern of DNA binding motifs is uniquely enriched in inaccessible chromatin at sites bound by these bHLH factors. These combined properties define a subclass of lineage-specific bHLH factors and provide context for their central roles in development and disease.

Preclinical characterization of tyrosine kinase inhibitor-based targeted therapies for neuroendocrine thyroid cancer.

Medullary thyroid carcinoma (MTC) is a slow growing neuroendocrine (NE) tumor for which few treatment options are available. Its incidence is rising and mortality rates have remained unchanged for decades. Increasing the repertoire of available treatments is thus crucial to manage MTC progression. Scarcity of patient samples and of relevant animal models are two challenges that have limited the development of effective non-surgical treatments. Here we use a clinically accurate mouse model of MTC to assess the effects and mode of action of the tyrosine kinase inhibitor (TKI) Vandetanib, one of only two drugs currently available to treat MTC. Effects on tumor progression, histopathology, and tumorigenic signaling were evaluated. Vandetanib blocked MTC growth through an anti-angiogenic mechanism. Furthermore, Vandetanib had an apparent anti-angiogenic effect in a patient MTC sample. Vandetanib displayed minimal anti-proliferative effects in vivo and in human and mouse MTC tumor-derived cells. Based on these results, we evaluated the second-generation TKI, Nintedanib, alone and in combination with the histone deacetylase (HDAC) inhibitor, Romidepsin, as potential alternative treatments to Vandetanib. Nintedanib showed an anti-angiogenic effect while Romidepsin decreased proliferation. Mechanistically, TKIs attenuated RET-, VEGFR2- and PI3K/AKT/FOXO signaling cascades. Nintedanib alone or in combination with Romidepsin, but not Vandetanib, inhibited mTOR signaling suggesting Nintedanib may have broader anti-cancer applicability. These findings validate the MTC mouse model as a clinically relevant platform for preclinical drug testing and reveal the modes of action and limitations of TKI therapies.

The Emerging Role of Cdk5 in Cancer.

Cdk5 is an atypical cyclin-dependent kinase that is well characterized for its role in the central nervous system rather than in the cell cycle. However Cdk5 has been recently implicated in the development and progression of a variety of cancers including breast, lung, colon, pancreatic, melanoma, thyroid and brain tumors. This broad pro-tumorigenic role makes Cdk5 a promising drug target for the development of new cancer therapies. Here we review the contribution of Cdk5 to molecular mechanisms that confer upon tumors the ability to grow, proliferate and disseminate to secondary organs, as well as resistance to chemotherapies. We subsequently discuss existing and new strategies for targeting Cdk5 and its downstream mechanisms as anti-cancer treatments.

Cdk5 Modulates Long-Term Synaptic Plasticity and Motor Learning in Dorsolateral Striatum.

The striatum controls multiple cognitive aspects including motivation, reward perception, decision-making and motor planning. In particular, the dorsolateral striatum contributes to motor learning. Here we define an approach for investigating synaptic plasticity in mouse dorsolateral cortico-striatal circuitry and interrogate the relative contributions of neurotransmitter receptors and intracellular signaling components. Consistent with previous studies, we show that long-term potentiation (LTP) in cortico-striatal circuitry is facilitated by dopamine, and requires activation of D1-dopamine receptors, as well as NMDA receptors (NMDAR) and their calcium-dependent downstream effectors, including CaMKII. Moreover, we assessed the contribution of the protein kinase Cdk5, a key neuronal signaling molecule, in cortico-striatal LTP. Pharmacological Cdk5 inhibition, brain-wide Cdk5 conditional knockout, or viral-mediated dorsolateral striatal-specific loss of Cdk5 all impaired dopamine-facilitated LTP or D1-dopamine receptor-facilitated LTP. Selective loss of Cdk5 in dorsolateral striatum increased locomotor activity and attenuated motor learning. Taken together, we report an approach for studying synaptic plasticity in mouse dorsolateral striatum and critically implicate D1-dopamine receptor, NMDAR, Cdk5, and CaMKII in cortico-striatal plasticity. Furthermore, we associate striatal plasticity deficits with effects upon behaviors mediated by this circuitry. This approach should prove useful for the study of the molecular basis of plasticity in the dorsolateral striatum.

Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis.

Memory enhancement by targeting Cdk5 regulation of NR2B.

Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor's cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers.

The role of Cdk5 in neuroendocrine thyroid cancer.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.

ß3 integrin interacts directly with GluA2 AMPA receptor subunit and regulates AMPA receptor expression in hippocampal neurons.

The integrins are transmembrane receptors for ECM proteins, and they regulate various cellular functions in the central nervous system. In hippocampal neurons, the ß3 integrin subtype is required for homeostatic synaptic scaling of AMPA receptors (AMPARs) induced by chronic activity deprivation. The surface level of ß3 integrin in postsynaptic neurons directly correlates with synaptic strength and the abundance of synaptic GluA2 AMPAR subunit. Although these observations suggest a functional link between ß3 integrin and AMPAR, little is known about the mechanistic basis for the connection. Here we investigate the nature of ß3 integrin and AMPAR interaction underlying the ß3 integrin-dependent control of synaptic AMPAR expression and thus synaptic strength. We show that ß3 integrin and GluA2 subunit form a complex in mouse brain that involves the direct binding between their cytoplasmic domains. In contrast, ß3 integrin associates with GluA1 AMPAR subunit only weakly, and, in a heterologous expression system, the interaction requires the coexpression of GluA2. Surprisingly, in hippocampal pyramidal neurons, expressing ß3 integrin mutants with either increased or decreased affinity for extracellular ligands has no differential effects in elevating excitatory synaptic currents and surface GluA2 levels compared with WT ß3 integrin. Our findings identify an integrin family member, ß3, as a direct interactor of an AMPAR subunit and provide molecular insights into how this cell-adhesion protein regulates the composition of cell-surface AMPARs.