Krishna Awasthi

Pentaazamacrocyclic Mn (II)-containing (MnPAM) superoxide dismutase mimetics can selectively modulate the effects of radiotherapy in tumors and normal tissues by enhancing oxidative stress in tumors and mitigating it in normal tissues. However, radiosensitizing effects in tumors are only achieved with stereotactic body radiotherapy-type dosing (≥7 Gy per fraction). These effects are limited at lower fractional doses that are clinically relevant for many tumors, including locally advanced rectal cancer. Here, we show that combining the MnPAM dismutase mimetic, rucosopasem manganese (GC4711), with pharmacological ascorbate significantly enhances radiosensitization to fractionated radiation doses relevant in colorectal cancer treatment (2–5 Gy) compared to either agent alone in multiple tumor models in vitro and significantly prolongs tumor growth delay in vivo. Mechanistically, enhanced radiosensitization with combination therapy is mediated by increased oxygen consumption and hydrogen peroxide generation. Despite having no effect on oxygen consumption in tumor cell media on its own, GC4711 significantly increases pharmacological ascorbate-induced oxygen consumption. Inducible catalase expression, or administration of exogenous catalase, attenuates the anti-cancer activity when combined. As predicted, increased peroxide fluxes with combination therapy enhanced postirradiation DNA damage and G2/M cell cycle accumulation. Importantly, treatment with pharmacological ascorbate and/or GC4711 had no significant effect on postirradiation clonogenic survival or DNA damage in non-malignant FHs74 enterocytes. These results support the hypothesis that adding pharmacological ascorbate to MnPAM dismutase mimetics selectively induces more peroxide-mediated radiosensitization than either agent alone in tumors without increasing radiosensitivity in cells treated with clinically relevant radiation fraction sizes used for radiotherapy of colorectal cancers.

Epidermal Growth Factor Receptor (EGFR) expression is upregulated in the majority of head and neck squamous cell carcinomas (HNSCC) and is associated with poor clinical outcomes. Unfortunately, the incorporation of EGFR inhibitors (EGFRIs) into the management of HNSCC has not improved long term survival rates in HNSCC patients despite high EGFR expression. Additionally, EGFR tyrosine kinase inhibitors (TKIs) in particular, have failed to show clinical benefit for HNSCC patients in clinical trials. Therefore, the identification and understanding of strategies that will improve the efficacy of EGFRIs may improve patient treatment and survival. Previous work in our lab has shown that HNSCC cells treated with erlotinib, a first-generation EGFR TKI, increased expression and release of the proinflammatory cytokine interleukin-1 alpha (IL-1α) resulting in inflammation and the development of drug resistance. The goal of this study is to determine if IL-1α-induced drug resistance is maintained across second, third and fourth generation EGFR TKIs.EGFR wildtype-positive HNSCC cells (FaDu, Cal-27 and SQ20B) were treated with the IC50 doses of erlotinib (first generation), afatinib (second generation), osimertinib (third generation), and silevertinib (fourth generation) EGFR TKIs for 48 h. Cell viability responses to EGFR TKIs were assessed using MTT assays. IL-1α and IL-6 cytokine expression was detected by ELISA and Western blot. Manipulation of IL-1 expression and signaling was performed using neutralizing antibodies to human IL-1α antibody (nIL-1α) and IL-1β (nIL-1β), and the human IL-1 receptor antagonist - anakinra. Results showed that all four EGFR TKIs triggered the release of IL-1α but not IL-1β from all 3 HNSCC cell lines. Additionally, all four EGFR TKIs increased IL-6 (IL-1α signaling endpoint) release, which was suppressed by nIL-1α and anakinra (but not nIL-1β) implying that IL-1α, rather than IL-1β, acts upstream of IL-6 activation. Overexpression of IL-1α in Cal-27 cells resulted in resistance to all four generations of EGFR TKIs. Lastly, erlotinib-resistant Cal 27 and SQ20B cells also demonstrated resistance to all other generations of EGFR TKIs. Collectively, these findings identify IL-1α expression as a central mediator of poor response and resistance to all EGFR TKIs via an EGFR mutation-independent mechanism. Hence, targeting IL-1α expression/signaling could be a promising strategy to improve long term tumor response to EGFR TKIs in HNSCC. Citation Format: Nafis Md Irfan, Ishrat Nourin Khan, Nurgul Koyuncu, Krishna Awasthi, Andrean L. Simons-Burnett. IL-1 alpha expression induces drug resistance to all generations of EGFR inhibitors in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7026.

Head and neck squamous cell carcinomas (HNSCCs) are extremely aggressive cancers that exhibit notable metabolic reprogramming and resistance to therapy. Phosphoglycerate mutase 1 (PGAM1), a glycolytic enzyme, has emerged as an important regulator of cancer cell metabolism and is highly expressed in HNSCCs compared to normal (untransformed) tissues. PGAM1 catalyzes the conversion of 3-phosphoglycerate (3PG) into 2-phosphoglycerate (2PG) which is a highly important step in glycolysis due to the importance of 2PG in serine biosynthesis. In contrast, PGAM1 inhibition results in decreased 2PG production and 3PG accumulation which suppresses both glycolysis and serine metabolism. More importantly, the accumulation of 3PG downregulates the pentose phosphate pathway (PPP) via deceased glucose-6-phosphate dehydrogenase (G6PD) which is necessary for optimal NADP+/NADPH production and cellular redox balance. Given the critical function of PGAM1, this enzyme may be promising as a potential target for treating HNSCC. Here we test if PGAM1 inhibition affects cell viability, NADP+/NADPH ratios, and DNA integrity in HNSCC cells in vitro. Gene expression levels of PGAM1 were compared between control and HNSCC patient groups, and survival analysis based on PGAM1 expression was conducted utilizing data from the TCGA database. PGAM1 was shown to be highly overexpressed in HNSCCs relative to healthy/control patients (P<0.001), with its expression linked to poorer survival (HR = 1.43). In addition, the PGAM1 expression level was evaluated based on human papillomavirus (HPV) subtype and found that PGAM1 is expressed significantly higher in HPV-negative patients compared to HPV-positive. The PGAM1 inhibitor - HKB99, was used to detect changes in cell viability via MTT assays in FaDu, Cal-27 and SQ20B HPV-negative HNSCC cell lines. HKB99 over a range of 0.5 - 64 µM for 48 hours decreased cell viability in a dose-dependent manner and IC50 concentrations were established which were 4 µM, 3.4 µM and 6.4 µM for FaDu, Cal-27 and SQ20B respectively. Treatment of the 3 cell lines with HKB99 at respective IC50 doses did not affect PGAM1 mRNA expression level using PCR analyses. However, protein expression of PGAM1 using western blot was decreased in treated cell lines compared to untreated cells. Moreover, HKB99 at respective IC50 doses increased levels of γ-H2AX, a marker for DNA double-strand breaks and significantly increased NADP+/NADPH ratios. Lastly, the antioxidant N-acetylcysteine (NAC) partially rescued the cytotoxic effect of HKB99 in all 3 cell lines using MTT and clonogenic assays. These findings suggest that inhibition of PGAM1 with HKB99 in HNSCC cells increases DNA damage, disrupts redox homeostasis, and triggers cell death. Taken together, our findings indicate that PGAM1 may represent a promising therapeutic target and may potentially sensitize HNSCCs to DNA-damaging therapies. Citation Format: Md Roman Mogal, Krishna Awasthi, Ishrat Nourin Khan, Andrean Simons-Burnett. Therapeutic targeting of phosphoglycerate mutase 1 disrupts cellular redox balance and promotes DNA damage in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1519.

ID 23594 Poster Board 249 Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Our recent studies demonstrated that exposure to pro-oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet-activating factor-receptor (PAFR), a G-protein coupled receptor present on various cell types, and acid sphingomyelinase (aSMase), an enzyme required for MVP biogenesis. Based upon this premise, we tested the hypothesis that topical application of gemcitabine will induce MVP generation in human and murine skin. Our ex vivo studies using human skin explants demonstrate that gemcitabine treatment results in MVP generation in a dose-dependent manner in a process blocked by PAFR antagonist and aSMase inhibitor. Importantly, gemcitabine-induced MVPs carry PAFR agonists. To confirm the mechanisms, we employed PAFR-expressing and deficient (Ptafr^-/-) mouse models as well as mice deficient in aSMase enzyme (Spmd1^-/-). Using genetic-based approaches, our studies demonstrated that gemcitabine-induced MVP release in WT mice was blunted in Ptafr^-/- and Spmd1^-/- mice. Overall, these findings indicate that pharmacological strategies to target the PAFR/aSMase pathway to block MVP release could be used as a promising approach for improving gemcitabine efficacy.

Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Our recent studies demonstrated that exposure to pro‐oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet‐activating factor‐receptor (PAFR), a G‐protein coupled receptor present on various cell types, and acid sphingomyelinase (aSMase), an enzyme required for MVP biogenesis. Based upon this premise, we tested the hypothesis that topical application of gemcitabine will induce MVP generation in human and murine skin. Our ex vivo studies using human skin explants demonstrate that gemcitabine treatment results in MVP generation in a dose‐dependent manner in a process blocked by PAFR antagonist and aSMase inhibitor. Importantly, gemcitabine‐induced MVPs carry PAFR agonists. To confirm the mechanisms, we employed PAFR‐expressing and deficient (Ptafr^−/−) mouse models as well as mice deficient in aSMase enzyme (Spmd1^−/−). Similar to the findings using pharmacologic tools, genetic‐based approaches demonstrate that gemcitabine‐induced MVP release in WT mice was blunted in Ptafr^−/− and Spmd1^−/− mice. These findings demonstrate a novel mechanism by which local chemotherapy can generate bioactive components as a bystander effect in a process that is dependent upon the PAFR‐aSMase pathway.

Most households may have leftover, unwanted, unused and expired (UUE) medicines. The present research aimed to analyze feasibility of implementation of medicine take-back in select communities in Nepal. Exploratory (i.e. feasibility) study was conducted among 400 adults from July 2017 to January 2018. Study sites and participants were selected by simple random sampling and respondents were interviewed about their awareness about medicine disposal, hazards and willingness to support take-back program using semi-structured questionnaire. Multinomial logistic regression analysis was applied to explore relationship of take-back related outcomes with the predictors. The P-value < 0.05 was statistically significant at 95% confidence level. Land pollution and effect on health of children was significantly related with inappropriate disposal of medicines such as site of disposal (P value < 0.01), river (P value, 0.02), garbage (P value, 0.04) and dumping site (P value, 0.01). Analysis of willingness to follow take-back program with the techniques of support showed significant relationship with the establishment of collection center and participation on seminar (P value < 0.01). Most participants were interested to support take-back, if implemented in their community but main constraint was the budget. Take-back concept could be initiated and implemented on government funding or other sources.

Background: Medications are not always completely consumed as purchased from pharmacy due to lack of awareness, forgetfulness. So, most households may have leftover, unwanted, unused and expired (UUE) medicines. They may lead to health hazards and environment pollution. The present study was aimed to analyze feasibility of implementation of medicine take-back in select communities of Nepal.Methods: Exploratory study was conducted among adults (total 400) in Kathmandu, Lalitpur, Kaski, Kanchanpur and Kailali districts from July 2017 to January 2018. Study sites and participants were selected by simple random sampling and respondents were interviewed about their awareness about medicine disposal techniques, possible hazards and their willingness to support take-back program using semi-structured questionnaire. Multinomial logistic regression analysis was applied to explore the relationship of various take-back related outcomes with the predictors. The p-value less than 0.05 was statistically significant at 95% confidence level. Results: The multinomial logistic regression analysis showed that there was significant relationship of health hazards (rashes and diarrhea) among the farmers, engineers, nurse and lawyers (p value <0.01 in each). Land pollution and effect on health of children was significantly related with the inappropriate disposal of medicines such as site of disposal (p value <0.01), river (p value 0.02), garbage (p value 0.04), and dumping site (p value 0.01). The analysis of the willingness to follow take-back program with the techniques of support showed significant relationship with the establishment of collection center and participation on seminar (p value <0.01). Conclusions: Most participants kept UUE medicines in home, disposed with household rubbish and flushed down the sink. They were interested to support take-back, if implemented in their community but the main constraint was the budget. Hence, take-back concept could be initiated and implemented on government funding or other external sources.

Background: Medications are not always completely consumed as purchased from pharmacy due to lack of awareness, forgetfulness. So, most households may have leftover, unwanted, unused and expired (UUE) medicines. They may lead to health hazards and environment pollution. The present study was aimed to analyze feasibility of implementation of medicine take-back in select communities of Nepal.Methods: Exploratory study was conducted among adults (total 400) in Kathmandu, Lalitpur, Kaski, Kanchanpur and Kailali districts from July 2017 to January 2018. Study sites and participants were selected by simple random sampling and respondents were interviewed about their awareness about medicine disposal techniques, possible hazards and their willingness to support take-back program using semi-structured questionnaire. Multinomial logistic regression analysis was applied to explore the relationship of various take-back related outcomes with the predictors. The p-value less than 0.05 was statistically significant at 95% confidence level. Results: The multinomial logistic regression analysis showed that there was significant relationship of health hazards (rashes and diarrhea) among the farmers, engineers, nurse and lawyers (p value Conclusions: Most participants kept UUE medicines in home, disposed with household rubbish and flushed down the sink. They were interested to support take-back, if implemented in their community but the main constraint was the budget. Hence, take-back concept could be initiated and implemented on government funding or other external sources.