Chimeric antigen receptor macrophages target and resorb amyloid plaques.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of ß amyloid (Aß) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aß-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting "reinforced CAR-Ms" have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aß in AD.

BAP1 promotes osteoclast function by metabolic reprogramming.

Treatment of osteoporosis commonly diminishes osteoclast number which suppresses bone formation thus compromising fracture prevention. Bone formation is not suppressed, however, when bone degradation is reduced by retarding osteoclast functional resorptive capacity, rather than differentiation. We find deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells (Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM osteoclasts fail to organize their cytoskeleton which is essential for bone degradation consequently increasing bone mass in both male and female mice. The deubiquitinase activity of BAP1 modifies osteoclast function by metabolic reprogramming. Bap1 deficient osteoclast upregulate the cystine transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11 controls cellular reactive oxygen species levels and redirects the mitochondrial metabolites away from the tricarboxylic acid cycle, both being necessary for osteoclast function. Thus, in osteoclasts BAP1 appears to regulate the epigenetic-metabolic axis and is a potential target to reduce bone degradation while maintaining osteogenesis in osteoporotic patients.

Initial clinical experience building a dual CT- and MR-guided adaptive radiotherapy program.

Introduction: Our institution was the first in the world to clinically implement MR-guided adaptive radiotherapy (MRgART) in 2014. In 2021, we installed a CT-guided adaptive radiotherapy (CTgART) unit, becoming one of the first clinics in the world to build a dual-modality ART clinic. Herein we review factors that lead to the development of a high-volume dual-modality ART program and treatment census over an initial, one-year period. Materials and Methods: The clinical adaptive service at our institution is enabled with both MRgART (MRIdian, ViewRay, Inc, Mountain View, CA) and CTgART (ETHOS, Varian Medical Systems, Palo Alto, CA) platforms. We analyzed patient and treatment information including disease sites treated, radiation dose and fractionation, and treatment times for patients on these two platforms. Additionally, we reviewed our institutional workflow for creating, verifying, and implementing a new adaptive workflow on either platform. Results: From October 2021 to September 2022, 256 patients were treated with adaptive intent at our institution, 186 with MRgART and 70 with CTgART. The majority (106/186) of patients treated with MRgART had pancreatic cancer, and the most common sites treated with CTgART were pelvis (23/70) and abdomen (20/70). 93.0% of treatments on the MRgART platform were stereotactic body radiotherapy (SBRT), whereas only 72.9% of treatments on the CTgART platform were SBRT. Abdominal gated cases were allotted a longer time on the CTgART platform compared to the MRgART platform, whereas pelvic cases were allotted a shorter time on the CTgART platform when compared to the MRgART platform. Our adaptive implementation technique has led to six open clinical trials using MRgART and seven using CTgART. Conclusions: We demonstrate the successful development of a dual platform ART program in our clinic. Ongoing efforts are needed to continue the development and integration of ART across platforms and disease sites to maximize access and evidence for this technique worldwide.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. EXPERIMENTAL DESIGN: We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. RESULTS: Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. CONCLUSIONS: These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

Chimeric Antigen Receptor Macrophages Target and Resorb Amyloid Plaques in a Mouse Model of Alzheimer's Disease.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). Several monoclonal antibodies targeting aggregated forms of beta amyloid (Aß), have been shown to reduce amyloid plaques and in some cases, mitigate cognitive decline in early-stage AD patients. We sought to determine if genetically engineered macrophages could improve the targeting and degradation of amyloid plaques. Chimeric antigen receptor macrophages (CAR-Ms), which show promise as a cancer treatment, are an appealing strategy to enhance target recognition and phagocytosis of amyloid plaques in AD. We genetically engineered macrophages to express a CAR containing the anti-amyloid antibody aducanumab as the external domain and the Fc receptor signaling domain internally. CAR-Ms recognize and degrade Aß in vitro and on APP/PS1 brain slices ex vivo; however, when injected intrahippocampally, these first-generation CAR-Ms have limited persistence and fail to reduce plaque load. We overcame this limitation by creating CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. These CAR-Ms have greater survival in the brain niche, and significantly reduce plaque load locally in vivo. These proof-of-principle studies demonstrate that CAR-Ms, previously only applied to cancer, may be utilized to target and degrade unwanted materials, such as amyloid plaques in the brains of AD mice.

Intrinsic tumor resistance to CAR T cells is a dynamic transcriptional state that is exploitable with low-dose radiation.

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement for hematologic malignancies, with some patients achieving long-term remission. However, the majority of treated patients still die of their disease. A consistent predictor of response is tumor quantity, wherein a higher disease burden before CAR T-cell therapy portends a worse prognosis. Focal radiation to bulky sites of the disease can decrease tumor quantity before CAR T-cell therapy, but whether this strategy improves survival is unknown. We find that substantially reducing systemic tumor quantity using high-dose radiation to areas of bulky disease, which is commonly done clinically, is less impactful on overall survival in mice achieved by CAR T cells than targeting all sites of disease with low-dose total tumor irradiation (TTI) before CAR T-cell therapy. This finding highlights another predictor of response, tumor quality, the intrinsic resistance of an individual patient's tumor cells to CAR T-cell killing. Little is known about whether or how an individual tumor's intrinsic resistance may change under different circumstances. We find a transcriptional "death receptor score" that reflects a tumor's intrinsic sensitivity to CAR T cells can be temporarily increased by low-dose TTI, and the timing of this transcriptional change correlates with improved in vivo leukemia control by an otherwise limited number of CAR T cells. This suggests an actionable method for potentially improving outcomes in patients predicted to respond poorly to this promising therapy and highlights that intrinsic tumor attributes may be equally or more important predictors of CAR T-cell response as tumor burden.

p38MAPKα Stromal Reprogramming Sensitizes Metastatic Breast Cancer to Immunotherapy.

Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.

CARD8 is an inflammasome sensor for HIV-1 protease activity.

HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to targeting immutable components are needed to clear HIV-1 infection. Here, we report that the caspase recruitment domain-containing protein 8 (CARD8) inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells before viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for the clearance of persistent HIV-1 infection.

Dendritic Cell Paucity Leads to Dysfunctional Immune Surveillance in Pancreatic Cancer.

Here, we utilized spontaneous models of pancreatic and lung cancer to examine how neoantigenicity shapes tumor immunity and progression. As expected, neoantigen expression during lung adenocarcinoma development leads to T cell-mediated immunity and disease restraint. By contrast, neoantigen expression in pancreatic ductal adenocarcinoma (PDAC) results in exacerbation of a fibro-inflammatory microenvironment that drives disease progression and metastasis. Pathogenic TH17 responses are responsible for this neoantigen-induced tumor progression in PDAC. Underlying these divergent T cell responses in pancreas and lung cancer are differences in infiltrating conventional dendritic cells (cDCs). Overcoming cDC deficiency in early-stage PDAC leads to disease restraint, while restoration of cDC function in advanced PDAC restores tumor-restraining immunity and enhances responsiveness to radiation therapy.

Overall Survival of Breast Cancer Patients With Locoregional Failures Involving Internal Mammary Nodes.

PURPOSE: Internal mammary node recurrence after definitive breast cancer treatment is poorly characterized, with limited data to guide clinical management. The aim of this study was to analyze the outcomes of patients with recurrent breast cancer involving internal mammary nodes to understand their natural history and determine prognostic factors associated with improved overall survival. METHODS AND MATERIALS: We performed a retrospective analysis of 553 patients with recurrent breast cancer and identified 161 patients with radiographic evidence of locoregional recurrence as a first event. A total of 67 patients (42%) were identified with internal mammary involvement. Median follow-up times were 76 months from date of initial diagnosis and 30 months from date of recurrence. RESULTS: Of the 67 patients identified with internal mammary node failures, 10 (15%) presented with isolated recurrence, 14 (21%) presented with other sites of locoregional disease, and 43 (64%) presented with concomitant distant metastases. Median overall survival was 2.5 years and significantly associated with extent of disease (P < .0001). On multivariable analysis, concomitant distant metastases, inflammatory breast cancer, and triple negative histologic type were associated with worse overall survival, whereas salvage radiation therapy was associated with improved overall survival. Among the 10 patients with isolated internal mammary node failures, median progression-free survival was 6.0 years and salvage therapy with surgery, radiation, and chemotherapy were associated with the best outcomes. CONCLUSIONS: Patients with isolated internal mammary node recurrences achieved long-term survival with aggressive therapies, and salvage radiation therapy was associated with improved survival.

CAR T-cell therapy for pancreatic cancer.

Chimeric antigen receptor (CAR) T-cell therapy utilizes genetic engineering to redirect a patient's own T cells to target cancer cells. The remarkable results in hematological malignancies prompted investigating this approach in solid tumors such as pancreatic cancer. The complex tumor microenvironment, stromal hindrance in limiting immune response, and expression of checkpoint blockade on T cells pose hurdles. Herein, we summarize the opportunities, challenges, and state of knowledge in targeting pancreatic cancer with CAR T-cell therapy.

Paxillin contracts the osteoclast cytoskeleton.

Osteoclastic bone resorption depends upon the cell's ability to organize its cytoskeleton via the αvß3 integrin and osteoclastogenic cytokines. Because paxillin associates with αvß3, we asked if it participates in skeletal degradation. Unlike deletion of other αvß3-associated cytoskeleton-regulating molecules, which impairs the cell's ability to spread, paxillin-deficient (Pax(-/-) ) osteoclasts, generated from embryonic stem cells, "superspread" in response to receptor activator of NF-κB ligand (RANKL) and form large, albeit dynamically atypical, actin bands. Despite their increased size, Pax(-/-) osteoclasts resorb bone poorly, excavating pits approximately one-third normal depth. Ligand-occupied αvß3 or RANKL promotes paxillin serine and tyrosine phosphorylation, the latter via cellular sarcoma (c-Src). The abnormal Pax(-/-) phenotype is rescued by wild-type (WT) paxillin but not that lacking its LD4 domain. In keeping with the appearance of mutant osteoclasts, WT paxillin, overexpressed in WT cells, contracts the cytoskeleton. Most importantly, the abnormal phenotype of Pax(-/-) osteoclasts likely represents failed RANKL-mediated delivery of myosin IIA to the actin cytoskeleton via the paxillin LD4 domain but is independent of tyrosine phosphorylation. Thus, in response to RANKL, paxillin associates with myosin IIA to contract the osteoclast cytoskeleton, thereby promoting its bone-degrading capacity.

Halofuginone prevents estrogen-deficient osteoporosis in mice.

Osteoporosis is characterized by enhanced activity of osteoclasts relative to that of osteoblasts. Thus, the principal means of treating the most common form of osteoporosis, namely that attending menopause, is inhibition of osteoclast formation or function. We have demonstrated that the inflammatory cytokine, IL-17, mediates estrogen-deficient osteoporosis, in mice, and that genetic blockade of its function prevents ovariectomy-induced bone loss. We herein report that the febrifugine derivative, halofuginone, a small molecule drug, reduces abundance of Th-17 cells in mice and prevents estrogen-deficient osteoporosis by diminishing bone resorption without impacting osteogenesis. In keeping with IL-17 mediating its osteoclastogenic effects by promoting RANK ligand expression by osteoblasts, halofuginone does not directly inhibit the bone resorptive cell. Thus, halofuginone, which is presently FDA-approved for treatment of scleroderma, is a candidate therapeutic for post-menopausal osteoporosis.

IL-17 mediates estrogen-deficient osteoporosis in an Act1-dependent manner.

Estrogen-deficient osteoporosis may be an inflammatory disorder and we therefore asked if IL-17 participates in its pathogenesis. Deletion of the principal IL-17 receptor (IL-17RA) protects mice from ovariectomy (OVX)-induced bone loss. Further supporting a central role of IL-17 in its pathogenesis, OVX-induced osteoporosis is prevented by a blocking antibody targeting the cytokine. IL-17 promotes osteoclastogenesis by stimulating RANK ligand (RANKL) expression by osteoblastic cells, mediated by the IL-17RA SEFIR/TILL domain. Estrogen deprivation, however does not enhance IL-17RA mRNA expression by osteoblasts or in bone, but augments that of Act1, an IL-17RA-interacting protein and signaling mediator. Similar to IL-17RA(-/-) mice, those lacking Act1 are protected from OVX-induced bone loss. Also mirroring IL-17RA-deficiency, absence of Act1 in osteoblasts, but not osteoclasts, impairs osteoclastogenesis via dampened RANKL expression. Transduction of WT Act1 into Act1(-/-) osteoblasts substantially rescues their osteoclastogenic capacity. The same construct, however, lacking its E3 ligase U-box or its SEFIR domain, which interacts with its counterpart in IL-17RA, fails to do so. Estrogen deprivation, therefore, promotes RANKL expression and bone resorption in association with upregulation of the IL-17 effector, Act1, supporting the concept that post-menopausal osteoporosis is a disorder of innate immunity.

Autophagy proteins regulate the secretory component of osteoclastic bone resorption.

Osteoclasts resorb bone via the ruffled border, whose complex folds are generated by secretory lysosome fusion with bone-apposed plasma membrane. Lysosomal fusion with the plasmalemma results in acidification of the resorptive microenvironment and release of CatK to digest the organic matrix of bone. The means by which secretory lysosomes are directed to fuse with the ruffled border are enigmatic. We show that proteins essential for autophagy, including Atg5, Atg7, Atg4B, and LC3, are important for generating the osteoclast ruffled border, the secretory function of osteoclasts, and bone resorption in vitro and in vivo. Further, Rab7, which is required for osteoclast function, localizes to the ruffled border in an Atg5-dependent manner. Thus, autophagy proteins participate in polarized secretion of lysosomal contents into the extracellular space by directing lysosomes to fuse with the plasma membrane. These findings are in keeping with a putative link between autophagy genes and human skeletal homeostasis.

Fyn promotes proliferation, differentiation, survival and function of osteoclast lineage cells.

c-Src and Lyn are the only Src family kinases (SFKs) with established activity in osteoclasts (OCs). c-Src promotes function via cytoskeletal organization of the mature resorptive cell while Lyn is a negative regulator of osteoclastogenesis. We establish that Fyn, another SFK, also impacts the OC, but in a manner distinctly different than c-Src and Lyn. Fyn deficiency principally alters cells throughout the osteoclastogenic process, resulting in diminished numbers of resorptive polykaryons. Arrested OC formation in the face of insufficient Fyn reflects reduced proliferation of precursors, in response to M-CSF and retarded RANK ligand (RANKL)-induced differentiation, attended by suppressed activation of the osteoclastogenic signaling molecules, c-Jun, and NF-κB. The anti-apoptotic properties of RANKL are also compromised in cells deleted of Fyn, an event mediated by increased Bim expression and failed activation of Akt. The defective osteoclastogenesis of Fyn-/- OCs dampens bone resorption, in vitro. Finally, while Fyn deficiency does not regulate basal osteoclastogenesis, in vivo, it reduces that stimulated by RANKL by ~2/3. Thus, Fyn is a pro-resorptive SFK, which exerts its effects by prompting proliferation and differentiation while attenuating apoptosis of OC lineage cells.