Paclitaxel is among the most widely used anticancer drugs and is known to cause a dose-limiting peripheral neurotoxicity, the initiating mechanisms of which remain unknown. Here, we identified the murine solute carrier organic anion–transporting polypeptide B2 (OATP1B2) as a mediator of paclitaxel-induced neurotoxicity. Additionally, using established tests to assess acute and chronic paclitaxel-induced neurotoxicity, we found that genetic or pharmacologic knockout of OATP1B2 protected mice from mechanically induced allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes. The function of this transport system was inhibited by the tyrosine kinase inhibitor nilotinib through a noncompetitive mechanism, without compromising the anticancer properties of paclitaxel. Collectively, our findings reveal a pathway that explains the fundamental basis of paclitaxel-induced neurotoxicity, with potential implications for its therapeutic management.
Alix F. Leblanc, Jason A. Sprowl, Paola Alberti, Alessia Chiorazzi, W. David Arnold, Alice A. Gibson, Kristen W. Hong, Marissa S. Pioso, Mingqing Chen, Kevin M. Huang, Vamsi Chodisetty, Olivia Costa, Tatiana Florea, Peter de Bruijn, Ron H. Mathijssen, Raquel E. Reinbolt, Maryam B. Lustberg, Lara E. Sucheston-Campbell, Guido Cavaletti, Alex Sparreboom, Shuiying Hu
T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who obtained a complete response following adoptive transfer of tumor infiltrating lymphocytes (TIL). Tumor exome sequencing surprisingly revealed less than 30 somatic mutations, including oncogenic BRAF V600E. Analysis of the specificity of TIL identified rare CD4 T cells specific for BRAFV600E and diverse CD8 T cells reactive to non-mutated self-antigens. These specificities increased in blood after TIL transfer and persisted long term suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC positive cells expressing the BRAF mutation. Therapy with TCR engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self and/or mutated tumor antigens in patients with BRAF mutated cancers.
Joshua R. Veatch, Sylvia M. Lee, Matthew Fitzgibbon, I-Ting Chow, Brenda Jesernig, Thomas Schmitt, Ying Ying Kong, Julia Kargl, A. McGarry Houghton, John A. Thompson, Martin McIntosh, William W. Kwok, Stanley R. Riddell
Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21lo B cells and plasmablasts. PD1 expression was higher in the CD21lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21hi cells. CCB induced proliferation in the CD21lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients.
Rituparna Das, Noffar Bar, Michelle Ferreira, Aaron M. Newman, Lin Zhang, Jithendra Kini Bailur, Antonella Bacchiocchi, Harriet Kluger, Wei Wei, Ruth Halaban, Mario Sznol, Madhav V. Dhodapkar, Kavita M. Dhodapkar
Nearly 50% of prostate cancers harbor gene fusions that lead to overexpression of the transcription factor ERG, while a mutually exclusive 10% of prostate cancers harbor recurrent mutations in the gene encoding the E3 ubiquitin ligase SPOP. Recent reports suggest that SPOP acts as a ubiquitin ligase for ERG and propose that ERG stabilization is the oncogenic effector of SPOP mutation. Here, we used human prostate cancer samples and showed that the vast majority of human SPOP-mutant cancers do not express ERG. Comparison of SPOP-mutant and ERG-fusion organoid models showed evidence of divergent, rather than common, transcriptional programs. Furthermore, expression of prostate cancer–associated SPOP mutations in genetically engineered mouse models of SPOP-mutant prostate cancer did not result in the expression of ERG protein in histologically normal prostate glands, high-grade prostatic intraepithelial neoplasia, invasive adenocarcinoma, or prostate organoids. In summary, we found no evidence that ERG is an effector of SPOP mutation in human prostate cancer or mouse models.
Jonathan Shoag, Deli Liu, Mirjam Blattner, Andrea Sboner, Kyung Park, Lesa Deonarine, Brian D. Robinson, Juan Miguel Mosquera, Yu Chen, Mark A. Rubin, Christopher E. Barbieri
Medulloblastoma, an aggressive cancer of the cerebellum, is among the most common pediatric brain tumors. Approximately one-third of medulloblastomas are associated with misactivation of the Hedgehog (Hh) pathway. GLI family zinc finger 2 (GLI2) coordinates the Hh transcriptional program; however, the GLI2 targets that promote cancer cell proliferation are unknown. Here, we incorporated a Gli2-EGFP allele into 2 different genetic mouse models of Hh-associated medulloblastoma. Hh signaling induced GLI2 binding to the Cdk6 promoter and activated Cdk6 expression, thereby promoting uncontrolled cell proliferation. Genetic or pharmacological inhibition of CDK6 in mice repressed the growth of Hh-associated medulloblastoma and prolonged survival through inhibition of cell proliferation. In human medulloblastoma, misactivation of Hh signaling was associated with high levels of CDK6, pointing to CDK6 as a direct transcriptional target of the Hh pathway. These results suggest that CDK6 antagonists may be a promising therapeutic approach for Hh-associated medulloblastoma in humans.
David R. Raleigh, Pervinder K. Choksi, Alexis Leigh Krup, Wasima Mayer, Nicole Santos, Jeremy F. Reiter
Primary immunodeficiencies are often monogenic disorders characterized by vulnerability to specific infectious pathogens. Here, we performed whole-exome sequencing of a patient with disseminated Mycobacterium tuberculosis, Streptococcus viridians bacteremia, and cytomegalovirus (CMV) viremia and identified mutations in 2 genes that regulate distinct IFN pathways. The patient had a homozygous frameshift deletion in IFNGR2, which encodes the signal transducing chain of the IFN-γ receptor, that resulted in minimal protein expression and abolished downstream signaling. The patient also harbored a homozygous deletion in IFNAR1 (IFNAR1*557Gluext*46), which encodes the IFN-α receptor signaling subunit. The IFNAR1*557Gluext*46 resulted in replacement of the stop codon with 46 additional codons at the C-terminus. The level of IFNAR1*557Gluext*46 mutant protein expressed in patient fibroblasts was comparable to levels of WT IFNAR1 in control fibroblasts. IFN-α–induced signaling was impaired in the patient fibroblasts, as evidenced by decreased STAT1/STAT2 phosphorylation, nuclear translocation of STAT1, and expression of IFN-α–stimulated genes critical for CMV immunity. Pretreatment with IFN-α failed to suppress CMV protein expression in patient fibroblasts, whereas expression of WT IFNAR1 restored IFN-α–mediated suppression of CMV. This study identifies a human IFNAR1 mutation and describes a digenic immunodeficiency specific to type I and type II IFNs.
Rodrigo Hoyos-Bachiloglu, Janet Chou, Catherine N. Sodroski, Abdallah Beano, Wayne Bainter, Magdalena Angelova, Eman Al Idrissi, Murad K. Habazi, Hamza Ali Alghamdi, Fahd Almanjomi, Mohamed Al Shehri, Nagi Elsidig, Morsi Alaa Eldin, David M. Knipe, Mofareh AlZahrani, Raif S. Geha
Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.
Michael G. Poulos, Pradeep Ramalingam, Michael C. Gutkin, Pierre Llanos, Katherine Gilleran, Sina Y. Rabbany, Jason M. Butler
The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associated Beclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.
Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre
P-element–induced wimpy testes (Piwi) proteins are known for suppressing retrotransposon activation in the mammalian germline. However, whether Piwi protein or Piwi-dependent functions occur in the mammalian soma is unclear. Contrary to germline-restricted expression, we observed that Piwi-like Miwi2 mRNA is indeed expressed in epithelial cells of the lung in adult mice and that it is induced during pneumonia. Further investigation revealed that MIWI2 protein localized to the cytoplasm of a discrete population of multiciliated airway epithelial cells. Isolation and next-generation sequencing of MIWI2-positive multiciliated cells revealed that they are phenotypically distinct from neighboring MIWI2-negative multiciliated cells. Mice lacking MIWI2 exhibited an altered balance of airway epithelial cells, demonstrating fewer multiciliated cells and an increase in club cells. During pneumococcal pneumonia, Miwi2-deficient mice exhibited increased expression of inflammatory mediators and increased immune cell recruitment, leading to enhanced bacterial clearance. Taken together, our data delineate MIWI2-dependent functions outside of the germline and demonstrate the presence of distinct subsets of airway multiciliated cells that can be discriminated by MIWI2 expression. By demonstrating roles for MIWI2 in airway cell identity and pulmonary innate immunity, these studies elucidate unanticipated physiological functions for Piwi proteins in somatic tissues.
Gregory A. Wasserman, Aleksander D. Szymaniak, Anne C. Hinds, Kazuko Yamamoto, Hirofumi Kamata, Nicole M.S. Smith, Kristie L. Hilliard, Claudia Carrieri, Adam T. Labadorf, Lee J. Quinton, Xingbin Ai, Xaralabos Varelas, Felicia Chen, Joseph P. Mizgerd, Alan Fine, Dónal O’Carroll, Matthew R. Jones
It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1–infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10–4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.
Gert U. Van Zyl, Mary Grace Katusiime, Ann Wiegand, William R. McManus, Michael J. Bale, Elias K. Halvas, Brian Luke, Valerie F. Boltz, Jonathan Spindler, Barbara Laughton, Susan Engelbrecht, John M. Coffin, Mark F. Cotton, Wei Shao, John W. Mellors, Mary F. Kearney
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