METTL14-mediated m6A epitranscriptomic modification contributes to chemotherapy-induced neuropathic pain by stabilizing GluN2A expression via IGF2BP2

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Abstract

Epigenetics is a biological process that modifies and regulates gene expression, affects neuronal function, and contributes to pain. However, the mechanism by which epigenetics facilitates and maintains chronic pain is poorly understood. We aimed to determine whether N6-methyladenosine (m6A) specifically modified by methyltransferase 14 (METTL14) alters neuronal activity and governs pain by sensitizing the GluN2A subunit of the N-methyl-D-aspartate receptor (NMDAR) in the dorsal root ganglion (DRG) neurons in a model of chemotherapy-induced neuropathic pain (CINP). Using dot blotting, immunofluorescence, gain/loss-of-function, and behavioral assays, we found that m6A levels were upregulated in L4–L6 DRG neurons in the CINP in a DBP/METT14-dependent manner, which was also confirmed in human DRGs. Blocking METTL14 reduced m6A methylation and attenuated pain hypersensitivity. Mechanistically, METTL14-mediated m6A modification facilitated the synaptic plasticity of DRG neurons by enhancing the GluN2A subunit of NMDAR, and inhibiting METTL14 blocked this effect. In contrast, overexpression of METTL14 upregulated m6A modifications, enhanced presynaptic NMDAR activity in DRG neurons, and facilitated pain sensation. Our findings reveal a previously unrecognized mechanism of METTL14-mediated m6A modification in DRG neurons to maintain neuropathic pain. Targeting these molecules may provide a new strategy for pain treatment.

Authors

Weicheng Lu, Xiaohua Yang, Weiqiang Zhong, Guojun Chen, Xinqi Guo, Qingqing Ye, Yixin Xu, Zhenhua Qi, Yaqi Ye, Jingyun Zhang, Yuge Wang, Xintong Wang, Shu Wang, Qiyue Zhao, Weian Zeng, Junting Huang, Huijie Ma, Jingdun Xie

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

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Abstract

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma – DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR-Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance whilst maintaining compliance and therapeutic benefit, we combined paxalisib with the anti-hyperglycemic drug, metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-sequencing, identifying changes in myelination and tumor immune microenvironment crosstalk. Together, we have identified a clinically relevant DIPG therapeutic combinatorial approach.

Authors

Ryan J. Duchatel, Evangeline R. Jackson, Sarah G. Parackal, Dylan Kiltschewskij, Izac J. Findlay, Abdul Mannan, Dilana E. Staudt, Bryce C. Thomas, Zacary P. Germon, Sandra Laternser, Padraic S. Kearney, M. Fairuz B. Jamaluddin, Alicia M. Douglas, Tyrone S. Beitaki, Holly P. McEwen, Mika L. Persson, Emily A. Hocke, Vaibhav Jain, Michael Aksu, Elizabeth E. Manning, Heather C. Murray, Nicole M. Verrills, Claire Xin Sun, Paul Daniel, Ricardo E. Vilain, David A. Skerrett-Byrne, Brett Nixon, Susan Hua, Charles E. de Bock, Yolanda Colino-Sanguino, Fatima Valdes-Mora, Maria Tsoli, David S. Ziegler, Murray J. Cairns, Eric H. Raabe, Nicholas A. Vitanza, Esther Hulleman, Timothy N. Phoenix, Carl Koschmann, Frank Alvaro, Christopher V. Dayas, Christopher L. Tinkle, Helen Wheeler, James R. Whittle, David D. Eisenstat, Ron Firestein, Sabine Mueller, Santosh Valvi, Jordan R. Hansford, David M. Ashley, Simon G. Gregory, Lindsay B. Kilburn, Javad Nazarian, Jason E. Cain, Matthew D. Dun

The CoREST repressor complex mediates phenotype switching and therapy resistance in melanoma

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Abstract

Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through non-mutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated to date. Here, we evaluate the CoREST repressor complex and the recently developed bivalent inhibitor, corin, within the context of melanoma phenotype plasticity and therapeutic resistance. We find that CoREST is a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells leads to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin reveals specific effects on histone marks connected to EMT-associated transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor (BRAFi)-resistant melanomas with corin promotes resensitization to BRAFi therapy. DUSP1 is consistently downregulated in BRAFi-resistant melanomas which is reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity can be recapitulated by the p38 MAPK inhibitor, BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial to patients with BRAFi-resistant melanoma.

Authors

Muzhou Wu, Ailish Hanly, Frederick Gibson, Robert Fisher, Samantha Rogers, Kihyun Park, Angelina Zuger, Kevin Kuang, Jay H. Kalin, Sarah Nocco, Matthew Cole, Amy Xiao, Filisia Agus, Adam Labadorf, Samuel Beck, Marianne Collard, Philip A. Cole, Rhoda M. Alani

HER2 heterogeneity and treatment response-associated profiles in HER2-positive breast cancer in the NCT02326974 clinical trial

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Abstract

BACKGROUND. HER2-targeting therapies have great efficacy in HER2-positive breast cancer, but resistance in part due to HER2 heterogeneity (HET) is a significant clinical challenge. We previously described that in a phase II neoadjuvant trastuzumab emtansine (T-DM1) and pertuzumab (T-DM1+P) clinical trial in early-stage HER2-positive breast cancer, none of the patients with HER2-HET tumors had pathologic complete response (pCR). METHODS. To investigate cellular and molecular differences among tumors according to HER2 heterogeneity and pCR, we performed RNA sequencing (RNA-seq) and ERBB2 FISH of 285 pre/post-treatment tumors from 129 patients in this T-DM1+P neoadjuvant trial. A subset of cases was also subject to Nanostring spatial digital profiling. RESULTS. Pre-treatment tumors from patients with pCR had the highest level of ERBB2 mRNA and ERBB signaling. HET was associated with no pCR, basal-like features, low ERBB2 expression yet high ERBB signaling sustained by activation of downstream pathway components. Residual tumors showed decreased HER2 protein levels and ERBB2 copy number heterogeneity and increased PI3K pathway enrichment and luminal features. HET tumors showed minimal treatment-induced transcriptomic changes compared to non-HET tumors. Immune infiltration correlated with pCR and HER2-HET status. CONCLUSION. Resistance mechanisms in HET and non-HET tumors are distinct. HER2-targeting antibodies have limited efficacy in HET tumors. Our results support the stratification of patients based on HET status and the use of agents that target downstream components of the ERBB signaling pathway in patients with HET tumors. TRIAL REGISTRATION. Clinicaltrials.gov NCT02326974. FUNDING. This study was funded by Roche and the National Cancer Institute.

Authors

Zheqi Li, Otto Metzger Filho, Giuseppe Viale, Patrizia dell'Orto, Leila Russo, Marie-Anne Goyette, Avni Kamat, Denise A. Yardley, Vandana Gupta Abramson, Carlos L. Arteaga, Laura M. Spring, Kami Chiotti, Carol Halsey, Adrienne G. Waks, Tari A. King, Susan C. Lester, Jennifer R. Bellon, Eric P. Winer, Paul T. Spellman, Ian E. Krop, Kornelia Polyak

The D84G mutation in STIM1 causes nuclear envelope dysfunction and myopathy in mice

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Abstract

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor located in the sarcoplasmic reticulum (SR) of skeletal muscle where it is best known for its role in store operated Ca2+ entry (SOCE). Genetic syndromes resulting from STIM1 mutations are recognized as a cause of muscle weakness and atrophy. Here, we focus on a gain of function mutation that occurs in humans and mice (STIM1+/D84G mice) where muscles exhibit constitutive SOCE. Unexpectedly, this constitutive SOCE did not affect global Ca2+ transients, SR Ca2+ content or excitation contraction coupling (ECC) and was therefore unlikely to underlie the reduced muscle mass and weakness observed in these mice. Instead, we demonstrate that the presence of D84G STIM1 in the nuclear envelope disrupts nuclear-cytosolic coupling causing severe derangement in nuclear architecture of STIM1+/D84G muscle, DNA damage and altered lamina A associated gene expression. Functionally, we found D84G STIM1 reduced the transfer of Ca2+ from the cytosol to the nucleus in myoblasts resulting in a reduction of [Ca2+]N. Taken together, we propose a novel role for STIM1 in the nuclear envelope that links Ca2+ signaling to nuclear stability in skeletal muscle.

Authors

Victoria Bryson, Chaojian Wang, Zirui Zhou, Kavisha Singh, Noah M. Volin, Eda Yildirim, Paul Rosenberg

MMR vaccination induces trained immunity via functional and metabolic reprogramming of γδ T cells

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Abstract

The measles, mumps and rubella (MMR) vaccine protects against all-cause mortality in children, but the immunological mechanisms mediating these effects are poorly known. We systematically investigated whether MMR can induce long-term functional changes in innate immune cells, a process termed trained immunity, that could at least partially mediate this heterologous protection. In a randomized placebo-controlled trial, 39 healthy adults received either the MMR vaccine or a placebo. By using single-cell RNA-sequencing, we found that MMR caused transcriptomic changes in CD14-positive monocytes and NK cells, but most profoundly in γδ T cells. Monocyte function was not altered by MMR vaccination. In contrast, the function of γδ T cells was markedly enhanced by MMR vaccination, with higher production of TNF and IFNγ, as well as upregulation of cellular metabolic pathways. In conclusion, we describe a trained immunity program characterized by modulation of γδ T cell function induced by MMR vaccination.

Authors

Rutger J. Röring, Priya A. Debisarun, Javier Botey-Bataller, Tsz Kin Suen, Ozlem Bulut, Gizem Kilic, Valerie A.C.M. Koeken, Andrei Sarlea, Harsh Bahrar, Helga Dijkstra, Heidi Lemmers, Katharina L. Gössling, Nadine Rüchel, Philipp N. Ostermann, Lisa Müller, Heiner Schaal, Ortwin Adams, Arndt Borkhardt, Yavuz Ariyurek, Emile J. de Meijer, Susan L. Kloet, Jaap ten Oever, Katarzyna Placek, Yang Li, Mihai G. Netea

mTORC1 controls murine postprandial hepatic glycogen synthesis via Ppp1r3b

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Abstract

In response to a meal, insulin drives hepatic glycogen synthesis to help regulate systemic glucose homeostasis. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-established insulin target and contributes to the postprandial control of liver lipid metabolism, autophagy, and protein synthesis. However, its role in hepatic glucose metabolism is less understood. Here, we used metabolomics, isotope tracing, and mouse genetics to define a role for liver mTORC1 signaling in the control of postprandial glycolytic intermediates and glycogen deposition. We show that mTORC1 is required for glycogen synthase activity and glycogenesis. Mechanistically, hepatic mTORC1 activity promotes the feeding-dependent induction of Ppp1r3b, a gene encoding a phosphatase important for glycogen synthase activity whose polymorphisms are linked to human diabetes. Re-expression of Ppp1r3b in livers lacking mTORC1 signaling enhances glycogen synthase activity and restores postprandial glycogen content. mTORC1-dependent transcriptional control of Ppp1r3b is facilitated by FOXO1, a well characterized transcriptional regulator involved in the hepatic response to nutrient intake. Collectively, we identify a role for mTORC1 signaling in the transcriptional regulation of Ppp1r3b and the subsequent induction of postprandial hepatic glycogen synthesis.

Authors

Kahealani Uehara, Won Dong Lee, Megan Stefkovich, Dipsikha Biswas, Dominic Santoleri, Anna E. Garcia Whitlock, William J. Quinn III, Talia N. Coopersmith, Kate Townsend Creasy, Daniel J. Rader, Kei Sakamoto, Joshua D. Rabinowitz, Paul M. Titchenell

A randomized first-in-human Phase 1 trial of differentially adjuvanted Pfs48/45 malaria vaccines in Burkinabé adults

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Abstract

BACKGROUND. Malaria transmission blocking vaccines aim to interrupt the transmission of malaria from one person to another. METHODS. The candidates, R0.6C and ProC6C, share the Plasmodium falciparum sexual stage antigen, Pfs48/45 “6C” domain. R0.6C utilizes the Glutamate Rich Protein (GLURP) as a carrier and ProC6C includes a second domain (Pfs230-Pro) and a short 36 amino acids CSP sequence. Healthy adults (n = 125) from a malaria endemic area of Burkina Faso were immunized with three intramuscular injections, four weeks apart, of 30 μg or 100 μg R0.6C or ProC6C each adsorbed to Alhydrogel adjuvant (AlOH) alone or in combination with Matrix-M (15 μg or 50 μg, respectively). The allocation was random and double blind for this Phase 1 trial. RESULTS. The vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of seven adverse events, mild to moderate in intensity and considered possibly related to the study vaccines were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 μg ProC6C-AlOH with Matrix-M, and 13/20 (65%) subjects in the group showed greater than 80% transmission reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA. CONCLUSIONS. All formulations were safe and well tolerated in a malaria endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation. TRIAL REGISTRATION. Pactr.org PACTR202201848463189. FUNDING. The study was funded by the European Union and Developing Countries Clinical Trials Partnership (Grant number RIA2018SV-2311).

Authors

B. Alfred Tiono, Jordan L. Plieskatt, Alphonse Ouedraogo, Ben Idriss Soulama, Kazutoyo Miura, Edith C. Bougouma, Mohammad Naghizadeh, Aissata Barry, Jean Baptiste B. Yaro, Sem Ezinmegnon, Noelie B. Henry, Ebenezer Ofori, Bright Adu, Susheel K. Singh, Augustin Konkobo, Karin Lövgren Bengtsson, Amidou Diarra, Cecilia Carnrot, Jenny M. Reimer, Amidou Z. Ouedraogo, Moussa Tienta, Carole A. Long, Issa N. Nebie, Issaka Sagara, Sodiomon B. Sirima, Michael Theisen

T antigen-specific CD8⁺ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma

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Abstract

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel Cell Polyomavirus, driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the non-responding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class-I haplotypes. We observed a broad T-cell recognition of T-Ags, including identification of 20 novel T-Ag-derived epitopes. Broadening of the T-Ag recognition profile and increased T-cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Authors

Ulla Kring Hansen, Candice D. Church, Ana Micaela Carnaz Simões, Marcus Svensson Frej, Amalie Kai Bentzen, Siri A. Tvingsholm, Jürgen C. Becker, Steven P. Fling, Nirasha Ramchurren, Suzanne L. Topalian, Paul T. Nghiem, Sine Reker Hadrup

p40 homodimers bridge ischemic tissue inflammation and heterologous alloimmunity in mice via IL-15 transpresentation

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Abstract

Virus-induced memory T cells often express functional cross-reactivity, or heterologous immunity, to other viruses and to allogeneic MHC molecules that is an important component of pathogenic responses to allogeneic transplants. During immune responses antigen-reactive naïve and central memory T cells proliferate in secondary lymphoid organs to achieve sufficient cell numbers to effectively respond whereas effector memory T cell proliferation occurs directly within the peripheral inflammatory microenvironment. Mechanisms driving heterologous memory T cell proliferation and effector function expression within peripheral tissues remain poorly understood. Here we dissected heterologous donor-reactive memory CD8 T cell proliferation and their effector functions following infiltration into heart allografts having low or high intensities of ischemic inflammation. Proliferation within both ischemic conditions requires p40 homodimer-induced IL-15 transpresentation by graft dendritic cells, but expression of effector functions mediating acute allograft injury occurs only in high-ischemic allografts. Transcriptional responses of heterologous donor-reactive memory CD8 T cells are distinct from donor antigen-primed memory CD8 T cells during early activation in allografts and at graft rejection. Overall, the results insights into mechanisms driving heterologous effector memory CD8 T cell proliferation and the separation between proliferation and effector function, that is dependent on the intensity of inflammation within the tissue microenvironment.

Authors

Hidetoshi Tsuda, Karen S. Keslar, William M. Baldwin III, Peter S. Heeger, Anna Valujskikh, Robert L. Fairchild