Clinical efficacy of plasmid encoding p62/SQSTM1 (Elenagen) in combination with gemcitabine in patients with platinum-resistant ovarian cancer: a randomized controlled trial.

Background: The purpose of this trial is to evaluate the safety and efficacy of ELENAGEN, a novel anticancer therapeutic DNA plasmid encoding p62/SQSTM1 protein, as an adjuvant to chemotherapy with gemcitabine (GEM) in patients with advanced platinum-resistant ovarian cancer. Methods: This open-label prospective randomized study with two arms. GEM (1000 mg/m2) on days 1 and 8 every 3 weeks was administered in both arms: in the Chemo arm (n = 20), GEM was the only treatment, and in the ELENAGEN arm (n = 20), GEM was supplemented with ELENAGEN (2.5 mg i.m. weekly). The primary endpoint was progression-free survival (PFS), and the secondary endpoint was safety. Antitumor activity was assessed by RECIST 1.1, and criteria safety was assessed according to NCI CTCAE version 5.0. Results: According to the cutoff data, the median follow-up was 13.8 months. There were no serious adverse events related to ELENAGEN treatment. The median PFS was 2.8 and 7.2 months in the Chemo and ELENAGEN arms, respectively (p Log-Rank = 0.03). Notably, at the time of cutoff, 9 patients (45%) in the ELENAGEN arm did not progress, with the longest PFS recorded thus far being 24 months. Subgroup analysis of patients in both arms demonstrated high efficacy of ELENAGEN in patients with worse prognostic factors: high pretreatment levels of CA125 and progression after platinum-free interval <3 months. Conclusions: The addition of ELENAGEN to gemcitabine is effective in patients with platinum-resistant ovarian cancer, including those with a worse prognosis. Clinical trial registration: https://www.clinicaltrials.gov/study/NCT05979298, identifier NCT05979298, 2023-08-07.

p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats.

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.

P62/SQSTM1 beyond Autophagy: Physiological Role and Therapeutic Applications in Laboratory and Domestic Animals.

Inflammation is the preceding condition for the development of mild and severe pathological conditions, including various forms of osteopenia, cancer, metabolic syndromes, neurological disorders, atherosclerosis, cardiovascular, lung diseases, etc., in human and animals. The inflammatory status is induced by multifarious intracellular signaling cascades, where cytokines, chemokines, arachidonic acid metabolites, adhesion molecules, immune cells and other components foster a "slow burn" at a local or systemic level. Assuming that countering inflammation limits the development of inflammation-based diseases, a series of new side-effects-free therapies was assessed in experimental and domestic animals. Within the targets of the drug candidates for quenching inflammation, an archetypal autophagic gear, the p62/sqstm1 protein, has currently earned attention from researchers. Intracellular p62 has been recently coined as a multi-task tool associated with autophagy, bone remodeling, bone marrow integrity, cancer progression, and the maintenance of systemic homeostasis. Accordingly, p62 can act as an effective suppressor of inflamm-aging, reducing oxidative stress and proinflammatory signals. Such an operational schedule renders this protein an effective watchdog for degenerative diseases and cancer development in laboratory and pet animals. This review summarizes the current findings concerning p62 activities as a molecular hub for cell and tissues metabolism and in a variety of inflammatory diseases and other pathological conditions. It also specifically addresses the applications of exogenous p62 (DNA plasmid) as an anti-inflammatory and homeostatic regulator in the treatment of osteoporosis, metabolic syndrome, age-related macular degeneration and cancer in animals, and the possible application of p62 plasmid in other inflammation-associated diseases.

P62/SQSTM1 enhances osteogenesis and attenuates inflammatory signals in bone marrow microenvironment.

Bone marrow-derived mesenchymal/stromal stem cells (MSCs) became a major focus of research since the anti-inflammatory features and the osteogenic commitment of these cells can prevent the inflamm-aging and various form of osteopenia in humans and animals. We previously showed that p62/SQSTM1 plasmid can prompt release of anti-inflammatory cytokines/chemokines by MSC when injected in adult mice. Furthermore, it can enhance osteoblastogenesis at the expense of adipogenesis and ameliorate bone density and bone remodeling. On the other hand, absence of p62 partially exhausted MSC pool caused expansion of fat cells within bone marrow and pro-inflammatory mediator's accumulation. Given the critical function of p62 as molecular hub of MSC dynamics, here, using MSCs from p62 knockout adult mice, we investigated the effect of this protein on MSC survival and bone-forming molecule cascades. We found that the main osteogenic routes are impaired in absence of p62. In particular, lack of p62 can suppress Smads activation, and Osterix and CREBs expression, thus significantly modifying the schedule of MSCs differentiation. MSCs obtained from p62-/- mice have also demonstrate an amplified NFκB/ Smad1/5/8 colocalization along with NFκB activation in the nucleus, which precludes Smads binding to target promoters. Considering the "teamwork" of TGFß, PTH and BMP2 on MSC homeostatic behavior, we consider that p62 exerts an essential role as a hub protein. Lastly, ex vivo pulsing p62-deficient MSCs, which then will be administered to a patient as a cell therapy, may be considered as a treatment for bone and bone marrow disorders.

The role of Bag3 in cell signaling.

Bag3 has been implicated in a wide variety of physiological processes from autophagy to aggresome formation and from cell transformation to survival. We argue that involvement of Bag3 in many of these processes is due to its distinct function in cell signaling. The structure of Bag3 suggests that it can serve as a scaffold that links molecular chaperones Hsp70 and small Hsps with components of a variety of signaling pathways. Major protein-protein interaction motifs of Bag3 that recruit components of signaling pathways are WW domain and PXXP motif that interacts with SH3-domain proteins. Furthermore, Hsp70-Bag3 appears to be a sensor of abnormal polypeptides during the proteotoxic stress. It also serves as a sensor of a mechanical force during mechanotransduction. Common feature of these and probably certain other sensory mechanisms is that they represent responses to specific kinds of abnormal proteins, i.e. unfolded filamin A in case of mechanotransduction or stalled translating polypeptides in case of sensing proteasome inhibition. Overall Hsp70-Bag3 module represents a novel signaling node that responds to multiple stimuli and controls multiple physiological processes.

HSP70s in Breast Cancer: Promoters of Tumorigenesis and Potential Targets/Tools for Therapy.

The high frequency of breast cancer worldwide and the high mortality among women with this malignancy are a serious challenge for modern medicine. A deeper understanding of the mechanisms of carcinogenesis and emergence of metastatic, therapy-resistant breast cancers would help development of novel approaches to better treatment of this disease. The review is dedicated to the role of members of the heat shock protein 70 subfamily (HSP70s or HSPA), mainly inducible HSP70, glucose-regulated protein 78 (GRP78 or HSPA5) and GRP75 (HSPA9 or mortalin), in the development and pathogenesis of breast cancer. Various HSP70-mediated cellular mechanisms and pathways which contribute to the oncogenic transformation of mammary gland epithelium are reviewed, as well as their role in the development of human breast carcinomas with invasive, metastatic traits along with the resistance to host immunity and conventional therapeutics. Additionally, intracellular and cell surface HSP70s are considered as potential targets for therapy or sensitization of breast cancer. We also discuss a clinical implication of Hsp70s and approaches to targeting breast cancer with gene vectors or nanoparticles downregulating HSP70s, natural or synthetic (small molecule) inhibitors of HSP70s, HSP70-binding antibodies, HSP70-derived peptides, and HSP70-based vaccines.

Response of a chemo-resistant triple-negative breast cancer patient to a combination of p62-encoding plasmid, Elenagen, and CMF chemotherapy.

Triple-negative breast cancers are often characterized by aggressive behavior and short clinical course once they become chemotherapy-resistant. We describe below a patient who has shown a response to combination of chemotherapy with Elenagen, a plasmid encoding p62. Elenagen was tested in a previous phase I/II study in patients with refractory solid tumors and shown to be safe. Also, plasmid ability to halt tumor progression and restore sensitivity to chemotherapy was found. Preclinical data supports effects on tumor grade and change the tumor's microenvironment in spontaneous canine breast cancers. We describe here a 48-year old female with triple-negative and BRCA1/2-negative breast cancer who had a primary resistance to chemotherapy and negative dynamics despite the use of multiple lines of treatments. Elenagen was applied intramuscularly at a dose of 1 mg weekly in combination with standard chemotherapy scheme CMF (cyclophosphamide, methotrexate, fluorouracil). In this patient we observed partial tumor regression (by 33%) and 19 weeks of progression-free survival. This first observed objective response to a combination of Elenagen with chemotherapy demonstrates that even in heavily pretreated chemo-resistant triple-negative tumor, the addition of Elenagen to a chemotherapy regimen can cause an objective response and increase in progression-free survival. Such a regimen is worthy of further study in a larger number of patients.

p62-DNA-encoding plasmid reverts tumor grade, changes tumor stroma, and enhances anticancer immunity.

Previously, we reported that the administration of a p62/SQSTM1-encoding plasmid demonstrates high safety and signs of clinical benefits for human cancer patients. The treatment also suppressed tumor growth and metastasis in dogs and mouse models. Here we investigated some mechanistic aspects of these effects. In mammary tumors bearing-dogs, i.m. injections of p62 plasmid reduced tumor sizes and their aggressive potential in 5 out of 6 animals, with one carcinoma switching to adenoma. The treatment increased levels of smooth muscle actin in stroma cells and type III collagen in the extracellular matrix, which correlate with a good clinical prognosis. The p62 treatment also increased the abundance of intratumoral T-cells. Because of the role of adaptive immunity cannot be tested in dogs, we compared the protective effects of the p62 plasmid against B16 melanoma in wild type C57BL/6J mice versus their SCID counterpart lacking lymphocytes. The plasmid was only protective in the wild type strain. Also, p62 plasmid amplified the anti-tumor effect of T-cell transfer from tumor-bearing animals to animals challenged with the same tumors. We conclude that the plasmid acts via re-modeling of the tumor microenvironment, making it more favorable for increased anti-cancer immunity. Thus, the p62-encoding plasmid might be a new adjuvant for cancer treatments.

p62 /SQSTM1 coding plasmid prevents age related macular degeneration in a rat model.

P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.

Cell Death and Survival Assays.

Heat shock proteins are well-known protectors from cell death. Cell death (in particular, apoptosis and necrosis) is accompanied by certain hallmarks manifested as specific alterations in cellular membranes, cytoplasm, nucleus, and mitochondria. Some of those hallmarks are easily detectable in situ and, therefore, they can be applied for the assessment of dying or dead cells. In turn, there are also signs of viable cells that include such features as normal functioning of their membranes and organelles, ability to proliferate, etc. This chapter describes several convenient methods for quantification of dead (apoptotic and necrotic) cells as well as methods for assessment of viable cells. We describe in detail methods of annexin V/propidium iodide (PI) staining, TUNEL assay, Hoechst/PI staining, caspase activation, MTS tetrazolium, lactate dehydrogenase (LDH) release, colony formation, and senescence assays, with the principles, advantages, and drawbacks of each technique.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

P62 plasmid can alleviate diet-induced obesity and metabolic dysfunctions.

A high-calorie diet (HCD) induces two mutually exacerbating effects contributing to diet-induced obesity (DIO): impaired glucose metabolism and increased food consumption. A link between the metabolic and behavioral manifestations is not well understood yet. We hypothesized that chronic inflammation induced by HCD plays a key role in linking together the two components of diet-induced pathology. Based on this hypothesis, we tested if a plasmid (DNA vaccine) encoding p62 (SQSTM1) would alleviate DIO including its metabolic and/or food consumption abnormalities. Previously we reported that injections of the p62 plasmid reduce chronic inflammation during ovariectomy-induced osteoporosis. Here we found that the p62 plasmid reduced levels of pro-inflammatory cytokines IL-1ß, IL-12, and INFγ and increased levels of anti-inflammatory cytokines IL-4, IL-10 and TGFß in HCD-fed animals. Due to this anti-inflammatory response, we further tested whether the plasmid can alleviate HCD-induced obesity and associated metabolic and feeding impairments. Indeed, p62 plasmid significantly reversed effects of HCD on the body mass index (BMI), levels of glucose, insulin and glycosylated hemoglobin (HbA1c). Furthermore, p62 plasmid partially restored levels of the satiety hormone, serotonin, and tryptophan, simultaneously reducing activity of monoamine oxidase (MAO) in the brain affected by the HCD. Finally, the plasmid partially reversed increased food consumption caused by HCD. Therefore, the administering of p62 plasmid alleviates both metabolic and behavioral components of HCD-induced obesity.

Anticancer Effects of Targeting Hsp70 in Tumor Stromal Cells.

The stress-induced chaperone protein Hsp70 enables the initiation and progression of many cancers, making it an appealing therapeutic target for development. Here, we show that cancer cells resistant to Hsp70 inhibitors in vitro remain sensitive to them in vivo, revealing the pathogenic significance of Hsp70 in tumor stromal cells rather than tumor cells as widely presumed. Using transgenic mouse models of cancer, we found that expression of Hsp70 in host stromal cells was essential to support tumor growth. Furthermore, genetic ablation or pharmacologic inhibition of Hsp70 suppressed tumor infiltration by macrophages needed to enable tumor growth. Overall, our results illustrate how Hsp70 inhibitors mediate the anticancer effects by targeting both tumor cells and tumor stromal cells, with implications for the broad use of these inhibitors as tools to ablate tumor-associated macrophages that enable malignant progression. Cancer Res; 76(20); 5926-32. ©2016 AACR.

Plasmid DNA-coding p62 as a bone effective anti-inflammatory/anabolic agent.

We recently reported that a DNA plasmid coding p62-SQSTM1 acts as an effective anti tumor vaccine against both transplantable mouse tumors and canine spontaneous mammary neoplasms. Here we report the unexpected finding that intramuscular delivery of p62 DNA exerts a powerful anti-osteoporotic activity in a mouse model of inflammatory bone loss (i.e, ovariectomy) by combining bone-sparing and osteo-synthetic effects. Notably, the suppression of osteoporosis by p62DNA was associated with a sharp down-regulation of master inflammatory cytokines, and up-regulation of endogenous p62 protein by bone-marrow stromal cells. The present data provide a solid rational to apply p62 DNA vaccine as a safe, new therapeutic for treatment of inflammatory related bone loss diseases.

Feasibility analysis of p62 (SQSTM1)-encoding DNA vaccine as a novel cancer immunotherapy.

Cancer immunotherapy is a thriving field, but its clinical achievements are modest so far. One of its major hurdles seems to be finding a feasible cancer antigen as a target for immune response. After many years of research, three major criteria for choice of tumor antigens emerged. An antigen should be: (i) immunogenic; (ii) essential for cancers cells (to avoid its loss through immunoediting), but dispensable for normal tissues to reduce the risk of toxicity, and (iii) overexpressed in tumors as compared to the normal tissues. Here we argue that p62 (SQSTM1), a protein involved in autophagy and signal transduction, fits all the above criteria and can be chosen as a novel cancer antigen. Accordingly, we carried out an extensive study and found antitumor and antimetastatic activity of p62-encoding DNA vaccine in five types of commonly used transplantable tumor models of mice and rats, and spontaneous tumors in several dogs. Given that toxicity of p62 vaccine was minimal, if any, we believe that p62-encoding vaccine merits further clinical development.

Pilot study of p62 DNA vaccine in dogs with mammary tumors.

Our previous data demonstrated profound anti-tumor and anti-metastatic effects of p62 (sqstm1) DNA vaccine in rodents with various types of transplantable tumors. Testing anti-cancer medicine in dogs as an intermediary step of translational research program provides two major benefits. First, clinical data collected in target animals is required for FDA/USDA approval as a veterinary anti-cancer drug or vaccine. It is noteworthy that the veterinary community is in need of novel medicine for the prevention and treatment of canine and feline cancers. The second more important benefit of testing anti-cancer vaccines in dogs is that spontaneous tumors in dogs may provide invaluable information for human trials. Here, we evaluated the effect(s) of p62 DNA vaccine on mammary tumors of dogs. We found that p62 DNA vaccine administered i.m. decreased or stabilized growth of locally advanced lesions in absence of its overall toxic effects. The observed antitumor activity was associated with lymphocyte infiltration and tumor encapsulation via fibrotic reaction. This data justifies both human clinical trials and veterinary application of p62 DNA vaccine.

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target.

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70.

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.

Proteasome failure promotes positioning of lysosomes around the aggresome via local block of microtubule-dependent transport.

Ubiquitinated proteins aggregate upon proteasome failure, and the aggregates are transported to the aggresome. In aggresomes, protein aggregates are actively degraded by the autophagy-lysosome pathway, but why targeting the aggresome promotes degradation of aggregated species is currently unknown. Here we report that the important factor in this process is clustering of lysosomes around the aggresome via a novel mechanism. Proteasome inhibition causes formation of a zone around the centrosome where microtubular transport of lysosomes is suppressed, resulting in their entrapment and accumulation. Microtubule-dependent transport of other organelles, including autophagosomes, mitochondria, and endosomes, is also blocked in this entrapment zone (E-zone), while movement of organelles at the cell periphery remains unaffected. Following the whole-genome small interfering RNA (siRNA) screen for proteins involved in aggresome formation, we defined the pathway that regulates formation of the E-zone, including the Stk11 protein kinase, the Usp9x deubiquitinating enzyme, and their substrate kinase MARK4. Therefore, upon proteasome failure, targeting of aggregated proteins of the aggresome is coordinated with lysosome positioning around this body to facilitate degradation of the abnormal species.