Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model.

Background: The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. Methods: To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. Results: The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. Conclusion: Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Concurrent Dexamethasone Limits the Clinical Benefit of Immune Checkpoint Blockade in Glioblastoma.

PURPOSE: Dexamethasone, a uniquely potent corticosteroid, is frequently administered to patients with brain tumors to decrease tumor-associated edema, but limited data exist describing how dexamethasone affects the immune system systemically and intratumorally in patients with glioblastoma (GBM), particularly in the context of immunotherapy. EXPERIMENTAL DESIGN: We evaluated the dose-dependent effects of dexamethasone when administered with programmed cell death 1 (PD-1) blockade and/or radiotherapy in immunocompetent C57BL/6 mice with syngeneic GL261 and CT-2A GBM tumors. Clinically, the effect of dexamethasone on survival was evaluated in 181 patients with isocitrate dehydrogenase (IDH) wild-type GBM treated with PD-(L)1 blockade, with adjustment for relevant prognostic factors. RESULTS: Despite the inherent responsiveness of GL261 to immune checkpoint blockade, concurrent dexamethasone administration with anti-PD-1 therapy reduced survival in a dose-dependent manner. Concurrent dexamethasone also abrogated survival following anti-PD-1 therapy with or without radiotherapy in immune-resistant CT-2A models. Dexamethasone decreased T-lymphocyte numbers by increasing apoptosis, in addition to decreasing lymphocyte functional capacity. Myeloid and natural killer cell populations were also generally reduced by dexamethasone. Thus, dexamethasone appears to negatively affect both adaptive and innate immune responses. As a clinical correlate, a retrospective analysis of 181 consecutive patients with IDH wild-type GBM treated with PD-(L)1 blockade revealed poorer survival among those on baseline dexamethasone. Upon multivariable adjustment with relevant prognostic factors, baseline dexamethasone administration was the strongest predictor of poor survival [reference, no dexamethasone; <2 mg HR, 2.16; 95% confidence interval (CI), 1.30-3.68; P = 0.003 and ≥2 mg HR, 1.97; 95% CI, 1.23-3.16; P = 0.005]. CONCLUSIONS: Our preclinical and clinical data indicate that concurrent dexamethasone therapy may be detrimental to immunotherapeutic approaches for patients with GBM.

Combined Targeting of the BRD4-NUT-p300 Axis in NUT Midline Carcinoma by Dual Selective Bromodomain Inhibitor, NEO2734.

NUT midline carcinoma (NMC) is a rare, aggressive subtype of squamous carcinoma that is driven by the BRD4-NUT fusion oncoprotein. BRD4, a BET protein, binds to chromatin through its two bromodomains, and NUT recruits the p300 histone acetyltransferse (HAT) to activate transcription of oncogenic target genes. BET-selective bromodomain inhibitors have demonstrated on-target activity in patients with NMC, but with limited efficacy. P300, like BRD4, contains a bromodomain. We show that combining selective p300/CBP and BET bromodomain inhibitors, GNE-781 and OTX015, respectively, induces cooperative depletion of MYC and synergistic inhibition of NMC growth. Treatment of NMC cells with the novel dual p300/CBP and BET bromodomain-selective inhibitor, NEO2734, potently inhibits growth and induces differentiation of NMC cells in vitro; findings that correspond with potentiated transcriptional effects from combined BET and p300 bromodomain inhibition. In three disseminated NMC xenograft models, NEO2734 provided greater growth inhibition, with tumor regression and significant survival benefit seen in two of three models, compared with a lead clinical BET inhibitor or "standard" chemotherapy. Our findings provide a strong rationale for clinical study of NEO2734 in patients with NMC. Moreover, the synergistic inhibition of NMC growth by CBP/p300 and BET bromodomain inhibition lays the groundwork for greater mechanistic understanding of the interplay between p300 and BRD4-NUT that drives this cancer.

LRRC15 Targeting in Soft-Tissue Sarcomas: Biological and Clinical Implications.

BACKGROUND: LRRC15 is a member of the LRR (leucine-rich repeat) superfamily present on tumor-associated fibroblasts (CAFs) and stromal cells. The expression of LRRC15 is upregulated by the pro-inflammatory cytokine TGFß. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) designed to target LRRC15, and which has shown significant anti-tumor activity in several tumor models. This is the first focused examination of LRRC15 expression and ABBV-085 activity in soft-tissue sarcomas (STS). METHODS: We analyzed the LRRC15 expression profile by immunohistochemistry in 711 STS cases, covering a broad spectrum of STS histologies and sub-classifications. In vivo experiments were carried out by using LRRC15-positive and LRRC15-negative patient-derived xenograft (PDX) models of STS. RESULTS: In contrast to patterns observed in epithelial tumors, LRRC15 was expressed not only by stromal cells but also by cancer cells in multiple subsets of STS with significant variations noted between histological subtypes. Overexpression of LRRC15 is positively correlated with grade and independently associated with adverse outcome. ABBV-085 has robust preclinical efficacy against LRRC15 positive STS patient-derived xenograft (PDX) models. CONCLUSION: We provide the first preclinical evidence that LRRC15 targeting with an antibody-drug conjugate is a promising strategy in LRRC15-positive STS. ABBV-085 is being evaluated in an ongoing clinical trial in STS and other malignancies.

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia.

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs.

Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.

Comparison of the Tastes of L-Alanine and Monosodium Glutamate in C57BL/6J Wild Type and T1r3 Knockout Mice.

Previous research showed that L-alanine and monosodium L-glutamate elicit similar taste sensations in rats. This study reports the results of behavioral experiments designed to compare the taste capacity of C57BL/6J wild type and T1r3- mice for these 2 amino acids. In conditioned taste aversion (CTA) experiments, wild-type mice exhibited greater sensitivity than knockout mice for both L-amino acids, although knockout mice were clearly able to detect both amino acids at 50 mM and higher concentrations. Generalization of CTA between L-alanine and L-glutamate was bidirectionally equivalent for both mouse genotypes, indicating that both substances elicited similar tastes in both genotypes. This was verified by the discrimination experiments in which both mouse genotypes performed at or near chance levels at 75 and 150 mM. Above 150 mM, discrimination performance improved, suggesting the taste qualities of the 2 L-amino acids are not identical. No differences between knockout and wild-type mice in discrimination ability were detected. These results indicate that while the T1r3 receptor is important for tasting L-alanine and L-glutamate, other receptors are also important for tasting these amino acids.

A conditioned aversion study of sucrose and SC45647 taste in TRPM5 knockout mice.

Previously, published studies have reported mixed results regarding the role of the TRPM5 cation channel in signaling sweet taste by taste sensory cells. Some studies have reported a complete loss of sweet taste preference in TRPM5 knockout (KO) mice, whereas others have reported only a partial loss of sweet taste preference. This study reports the results of conditioned aversion studies designed to motivate wild-type (WT) and KO mice to respond to sweet substances. In conditioned taste aversion experiments, WT mice showed nearly complete LiCl-induced response suppression to sucrose and SC45647. In contrast, TRPM5 KO mice showed a much smaller conditioned aversion to either sweet substance, suggesting a compromised, but not absent, ability to detect sweet taste. A subsequent conditioned flavor aversion experiment was conducted to determine if TRPM5 KO mice were impaired in their ability to learn a conditioned aversion. In this experiment, KO and WT mice were conditioned to a mixture of SC45647 and amyl acetate (an odor cue). Although WT mice avoided both components of the stimulus mixture, they avoided SC45647 more than the odor cue. The KO mice also avoided both stimuli, but they avoided the odor component more than SC45647, suggesting that while the KO mice are capable of learning an aversion, to them the odor cue was more salient than the taste cue. Collectively, these findings suggest the TRPM5 KO mice have some residual ability to detect SC45647 and sucrose, and, like bitter, there may be a TRPM5-independent transduction pathway for detecting these substances.

Double P2X2/P2X3 purinergic receptor knockout mice do not taste NaCl or the artificial sweetener SC45647.

The P2X ionotropic purinergic receptors, P2X2 and P2X3, are essential for transmission of taste information from taste buds to the gustatory nerves. Mice lacking both P2X2 and P2X3 purinergic receptors (P2X2/P2X3(Dbl-/-)) exhibit no taste-evoked activity in the chorda tympani and glossopharyngeal nerves when stimulated with taste stimuli from any of the 5 classical taste quality groups (salt, sweet, sour, bitter, and umami) nor do the mice show taste preferences for sweet or umami, or avoidance of bitter substances (Finger et al. 2005. ATP signaling is crucial for communication from taste buds to gustatory nerves. Science. 310[5753]:1495-1499). Here, we compare the ability of P2X2/P2X3(Dbl-/-) mice and P2X2/P2X3(Dbl+/+) wild-type (WT) mice to detect NaCl in brief-access tests and conditioned aversion paradigms. Brief-access testing with NaCl revealed that whereas WT mice decrease licking at 300 mM and above, the P2X2/P2X3(Dbl-/-) mice do not show any change in lick rates. In conditioned aversion tests, P2X2/P2X3(Dbl-/-) mice did not develop a learned aversion to NaCl or the artificial sweetener SC45647, both of which are easily avoided by conditioned WT mice. The inability of P2X2/P2X3(Dbl-/-) mice to show avoidance of these taste stimuli was not due to an inability to learn the task because both WT and P2X2/P2X3(Dbl-/-) mice learned to avoid a combination of SC45647 and amyl acetate (an odor cue). These data suggest that P2X2/P2X3(Dbl-/-) mice are unable to respond to NaCl or SC45647 as taste stimuli, mirroring the lack of gustatory nerve responses to these substances.

Behavioral studies of umami: tales told by mice and rats.

Psychophysical research with rats and mice has been instrumental in understanding umami taste transduction and perception. Although early studies suggested that an NMDA-like receptor detected substances that elicit an umami taste, studies using behavioral methods with both rats and mice indicate that the picture is much more complex. When the G protein-coupled receptor T1R1+T1R3 was discovered, it was believed to be the umami receptor and a more broadly tuned L-amino acid receptor. However, since then a number of behavioral studies, like molecular and physiological studies, report evidence that other receptors may contribute to umami taste. For example, T1R3 knockout mice (KO) have only slightly elevated detection thresholds for monosodium glutamate (MSG) and L-alanine. In conditioned taste aversion studies, T1R3 KO mice show bidirectional generalization of the aversion between MSG and L-alanine, suggesting that these substances have similar tastes. However, these KO mice can discriminate between the tastes of the two substances, indicating other receptors also respond to these amino acids. (RS)-alpha-cycloprophy-4-phosphonophenylglycine (CPPG), a potent mGluR4 antagonist, decreases an aversion to MSG in rats while increasing the strength of generalization of the aversion to L-arginine or L-serine. These behavioral studies suggest that glutamate can activate several putative receptors, most notably T1R1+T1R3 and taste-mGluR4, and possibly NMDA-like receptors or taste-mGluR1. These receptors generate similar but not identical sensations which, when combined, form a complex perception identified as umami. Further, these studies suggest that afferent signaling from T1R1+T1R3 and taste-mGluR4 likely combine to generate the taste sensations associated with other L-amino acids.