Gaucher disease (GD) is an autosomal recessive disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) remains the standard of care for non-neuropathic GD patients, its high cost significantly limits accessibility. To enhance production efficiency, we developed a lentiviral system encoding a codon-optimized GCase gene driven by the human elongation factor 1a (hEF1α) promoter for stable production in human cell lines. A functional lentiviral vector, LV_EF1α_GBA_Opt, was generated at a titer of 7.88 × 108 LV particles/mL as determined by qPCR. Six transduction cycles were performed at a multiplicity of infection of 30–50. The transduced heterogeneous human cell population showed GCase-specific activity of 307.5 ± 53.49 nmol/mg protein/h, which represents a 3.21-fold increase compared to wild-type 293FT cells (95.58 ± 16.5 nmol/mg protein/h). Following single-cell cloning, two clones showed specific activity of 763.8 ± 135.1 and 752.0 ± 152.1 nmol/mg/h (clones 15 and 16, respectively). These results show that codon optimization, a lentiviral delivery system, and clonal selection together enable the establishment of stable human cell lines capable of producing high levels of biologically active, synthetic recombinant GCase in vitro. Further studies are warranted for the functional validation in GD patient-derived fibroblasts and animal models.

Hematopoietic stem cells (HSCs) play a crucial role in generating all blood cell types, vital for a functional immune system and oxygen transport. Maintaining this balance throughout life involves a tight regulation of self-renewal, differentiation, and quiescence, influenced by both intrinsic and extrinsic signals. Although the influence of many HSC progeny on HSC decisions is known, the role of mast cells (MCs) has remained unexplored. MCs, known for their immunomodulatory functions through secretion of various factors, including histamine, present a novel avenue for understanding HSC regulation. In this study, we uncover a novel role for MC-derived histamine in modulating HSC behavior. Our hypothesis posits that MCs act as negative regulators of HSCs. We observed that genetically MC-deficient “SASH” mice exhibit increased hematopoietic output and bone marrow (BM) HSCs, characterized by an enhanced quiescent signature that increases chemoresistance. The SASH microenvironment also shows elevated frequencies of HSC-supportive cell types and increased expression of genes conducive to HSC maintenance, providing a functional advantage when wild-type BM is transplanted into this microenvironment. Moreover, we found that the genetic loss of MCs correlates with lower serum histamine levels in SASH mice, and the augmented hematopoietic phenotype can be reversed by administering exogenous histamine. Subsequent experiments with US Food and Drug Administration (US FDA)-approved antihistamines in wild-type mice revealed that cetirizine, an H1R inverse agonist, notably increased HSC frequency in the BM. We have also shown that cetirizine treatment posttransplant leads to an accelerated HSC and peripheral blood recovery. Overall, our findings highlighted MCs as negative regulators of HSCs, laying the groundwork for future studies to unravel the underlying mechanisms and explore the therapeutic potential of cetirizine.

In cancer cells, chromatin replication is often misregulated in part due to mutations in oncogenes and tumor suppressor genes, leading to increased replication stress and genomic instability. While this instability drives cancer progression, it also makes cancer cells more dependent on genome maintenance pathways, presenting a therapeutic target. A promising strategy is to exacerbate replication stress, causing lethal genome instability. We have developed a sensitive reporter to monitor replication stress via single-stranded DNA at stressed forks. Our earlier study identified Tousled-like kinases (TLKs) crucial for chromatin assembly during genome replication and discovered TLK inhibitors to treat cancers through increasing replication stress. Additionally, we recently uncovered histone deacetylase 8 (HDAC8) inhibitors that disrupt chromatin replication and compromise genome integrity, particularly when combined with checkpoint kinase inhibitors. In this presentation, I will show you how HDAC8 behaves as caretakers of genetic and epigenetic integrity and how we targeted replication stress to create cancer-specific synergistic vulnerabilities. Zih-Yao Yu, Wei-Jan Huang, Stanton Gerson, Sung-Bau Lee. HDAC8 as a critical regulator of cancer cell genome stability: therapeutic implications [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 1475.

ABSTRACTHematopoietic stem cells (HSCs) are essential for generating all blood cell types and maintaining immune function and oxygen transport. This requires tight regulation of self-renewal, differentiation, and quiescence, driven by intrinsic and extrinsic signals. While the influence of many HSC progeny on HSC decisions are recognized, the role of mast cells (MCs) remain understudied. MCs are known for their immunomodulatory functions through the secretion of factors such as histamine and could offer new insights into HSC regulation. In this study, we describe a novel role for MC-derived histamine in modulating HSC behavior. We observed that genetically MC-deficient “SASH” mice exhibit increased hematopoietic output and bone marrow (BM) HSCs, characterized by an enhanced quiescent signature that increases resistance to myeloablative chemotherapy. The SASH microenvironment also contained increased frequencies of HSC-supportive cell types and expression of genes conducive to HSC maintenance, which together accelerated HSC engraftment when wild-type BM was transplanted into SASH recipients. Moreover, we found lower serum histamine levels in SASH mice, and that the enhanced hematopoietic phenotype observed in these mice could be reversed by administering exogenous histamine. Subsequent experiments with FDA-approved antihistamines in wild-type mice revealed that cetirizine, an H1R inverse agonist, notably increased HSC frequency in the BM. Overall, our findings implicate MCs are negative regulators of HSC function. This lays the groundwork for future studies to elucidate the underlying mechanisms and explore the therapeutic potential of modulating histamine signaling to promote hematopoiesis.

12073 Background: The utilization of cannabis is increasing among patients with cancer; however, limited information exists about PMI with cannabis during treatment for cancer. Methods: An NCI-funded cross-sectional survey study was conducted at a comprehensive cancer center among patients undergoing anticancer therapy. Subjects completed a survey about utilization of medications including supplements and cannabis – specifically reasons for use and type of cannabis. All patients actively taking any cannabis were identified and included for analysis. Lexicomp and Natural Medicines Comprehensive Database interaction software were used to determine PMIs. Results: A total of 61 patients (19.5%) were identified from 313 participants as actively using a form of cannabis. Subjects had a mean age of 58 (SD±13.3) and mostly self-identified as being White (70%) with one-fifth being Black (21%). The most common types of cancer were lung (18%), breast (15%), lymphoma (12%) and colorectal (12%). The most prevalent modes of cannabis intake were smoking (36%), gummies (33%), and oil/tincture/liquid (16%). The most common reasons for cannabis use included insomnia (46%), pain (41%), and mood (39%). The median number of medications used were 6 (range, 1-16) and supplements (including cannabis) were 3 (range, 1-14). A total of 1157 PMIs and 569 unique PMIs were identified among active cannabis users. The most common PMIs were prescription-supplement interactions (31.7%) and prescription-cannabis interactions (27%). Nearly two-thirds of the PMIs (61%) were labelled as moderate, followed by major interactions (18%). Among these patients, there were 412 cannabis interactions (36% of PMIs) with 124 being unique. Of these cannabis PMIs, 71% were interactions with non-anticancer prescription medications, 17% with over-the-counter medications, and 10% with anticancer agents. Most of the cannabis PMIs were categorized as moderate (87%) and major (12%) severity, and the level of evidence of these PMIs were rated as poor (60%). The most common cannabis-related PMIs were with acetaminophen (9.5%), dexamethasone (8%), and ondansetron (6.3%). The most common chemotherapy PMIs were with paclitaxel (4%) and doxorubicin (2%). Conclusions: This is one of the first studies to assess cannabis-related drug interactions in patients receiving cancer treatment, and results indicates a significant proportion of cannabis users are at risk for moderate to major PMIs including with chemotherapy.[Table: see text]

The elevated level of replication stress is an intrinsic characteristic of cancer cells. Targeting the mechanisms that maintain genome stability to further increase replication stress and thus induce severe genome instability has become a promising approach for cancer treatment. Here, we identify histone deacetylase 8 (HDAC8) as a drug target whose inactivation synergized with the inhibition of checkpoint kinases to elicit substantial replication stress and compromise genome integrity selectively in cancer cells. We showed that simultaneous inhibition of HDAC8 and checkpoint kinases led to extensive replication fork collapse, irreversible cell-cycle arrest, and synergistic vulnerability in various cancer cells. The efficacy of the combination treatment was further validated in patient tumor-derived organoid (PDO) and xenograft mouse (PDX) models, providing important insights into patient-specific drug responses. Our data revealed that HDAC8 activity was essential for reducing the acetylation level of structural maintenance of chromosomes protein 3 (SMC3) ahead of replication forks and preventing R loop formation. HDAC8 inactivation resulted in slowed fork progression and checkpoint kinase activation. Our findings indicate that HDAC8 guards the integrity of the replicating genome, and the cancer-specific synthetic lethality between HDAC8 and checkpoint kinases provides a promising replication stress-targeting strategy for treating a broad range of cancers.

BACKGROUND:This study characterizes patient and health-care professional perspectives regarding medical cannabis use at a National Cancer Institute-Designated Cancer Center. Data evaluated included the prevalence and patterns of and reasons for cannabis use. METHODS:Patients with cancer undergoing treatment were recruited into a cross-sectional survey as part of a national National Cancer Institute-funded effort. Participants completed a survey about cannabis use, reasons for use, and types of cannabis. A health-care professional survey was also conducted to explore perspectives regarding patients' use of cannabis. RESULTS:A total of 313 patients with cancer (mean [SD] age = 60.7 [12.8] years) completed the survey (43% response rate) between 2021 and 2022. Of the respondents, 58% were female; identified as White (61%) and Black (23%); and had diverse cancer diagnoses. Nearly half of respondents (43%) had previously used cannabis, one-quarter (26%) had used cannabis since their cancer diagnosis, and almost 1 in 6 (17%) were actively using cannabis at the time of survey completion. The most common modes of ingestion were gummies (33%) and smoking (30%). The most commonly reported reasons for use were insomnia (46%), pain (41%), and mood (39%). For the 164 health-care professionals who completed the survey (25% response rate), the majority agreed that cannabis use (72%) is safe and beneficial for patients (57%). Four in 10 (39%) health-care professionals felt comfortable providing guidance to patients about cannabis use; however, only 1 in 8 (13%) felt knowledgeable about the topic of cannabis. CONCLUSIONS:Approximately one-sixth of patients with cancer receiving treatment actively use cannabis for management of various cancer symptoms. Perceptions about cannabis use and education varied widely among health-care professionals.

Objective. The neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR) demonstrate good diagnostic accuracy in distinguishing lung cancer patients from healthy individuals, primarily in HIV-negative populations. We determined the sensitivity (Se), specificity (Sp), and area under the curve (AUC) of the NLR and PLR in discriminating between people living with HIV (PLWH) with and without lung cancer. Methods. This is a comparative analysis of secondary data. Cases were PLWH with lung cancer from a retrospective cohort treated at the Uganda Cancer Institute. Controls were unmatched PLWH without lung cancer who were randomly selected from three HIV clinics in Uganda. Se, Sp, and AUC analysis and determination of optimal cutoffs were performed using receiver operating characteristic (ROC) curves. Results. Of 115 PLWH (18 cases and 97 controls), 83 (72.2%) were female, 110 (95.7) were on ART, and the median (IQR) age was 46 (38–51) years. The median (IQR) NLR was higher among cases than controls (3.53 (3.14–7.71) vs. 0.92 (0.67–1.09), p <0.001 ). Similarly, the PLR was higher among cases than controls (237.5 (177.8–361.6) vs. 123.6 (100.6–155.4), p=0.001 ). At a cutoff of 2.44, the respective Se, Sp, and AUC of the NLR were 87.5% (95% CI: 61.7%–98.4%), 100% (95% CI: 96.2%–100%), and 0.94 (95% CI: 0.85–1.00, p <0.001 ). Similarly, the respective Se, Sp, and AUC for the PLR were 75% (95% CI: 47.6%–92.7%), 87.2% (95% CI: 78.8%–93.2%), and 0.81 (95% CI: 0.70–0.93, p <0.001 ) at a cutoff of 196.3. Conclusion. The NLR and PLR discriminated PLWH with and without lung cancer and could be useful in PLWH with respiratory symptoms in whom lung cancer can easily be misdiagnosed as other lung pathology.

Hematopoietic stem cells (HSCs) play a crucial role in generating all blood cell types, vital for a functional immune system and oxygen transport. Maintaining this balance throughout life involves a tight regulation of self-renewal, differentiation, and quiescence, influenced by both intrinsic and extrinsic signals. While the influence of many HSC progeny on HSC decisions is known, the role of mast cells (MCs) has remained unexplored. MCs, known for their immunomodulatory functions through secretion of various factors including histamine, present a novel avenue for understanding HSC regulation. In this study, we uncover a novel role for MC-derived histamine in modulating HSC behavior. Our hypothesis posits that MCs act as negative regulators of HSCs. We observed that genetically MC-deficient “SASH” mice exhibit increased hematopoietic output and bone marrow (BM) HSCs, characterized by an enhanced quiescent signature that increases chemoresistance. The SASH microenvironment also shows elevated frequencies of HSC-supportive cell types and increased expression of genes conducive to HSC maintenance, providing a functional advantage when wild-type BM is transplanted into this microenvironment. Moreover, we found that the genetic loss of MCs correlates with lower serum histamine levels in SASH mice, and the augmented hematopoietic phenotype can be reversed by administering exogenous histamine. Subsequent experiments with FDA-approved antihistamines in wild-type mice revealed that cetirizine, an H1R inverse agonist, notably increased HSC frequency in the BM. Overall, our findings highlight MCs as negative regulators of HSCs, laying the groundwork for future studies to unravel the underlying mechanisms and explore the therapeutic potential of cetirizine.

Background: Gaucher disease (GD) is a rare lysosomal storage disease caused by pathogenic variants in the glucocerebrosidase gene (GBA1) resulting in a markedly decreased activity of the lysosomal enzyme b-glucocerebrosidase (GCase). Enzyme replacement therapy is the gold standard for treating GD patients. Aim: Here, we investigate whether in silico molecular evolution can be combined with synthetic biology and gene therapy for the development of a new recombinant enzyme with higher activity. Materials and methods: We developed novel GBA variants by introducing nonsynonymous mutations into the GBA gene obtained using in silico molecular evolution. These GBA variants are denoted as GBA-7, GBA-8, GBA-9 and GBA-12 and were cloned into lentiviral plasmids using synthetic biology approaches. Additionally, we obtained the wild-type GBA sequence from Homo sapiens, which is labeled GBA-Opt. In addition, we also aim to explore the differences in the transcriptional regulation using two different promoters: the CMV and the human elongation factor 1A (hEF1A). As a control, we generated a construct that expresses the green fluorescent protein (GFP) gene under control of the same promoters. The characterization of these DNA constructs was performed using 293FT human cell line. We generated a total of 166 293FT cell lines with transient production of GBA (n = 100), GBA + GFP (n = 40), GFP (n = 8), and included virgin cells (n = 18). Out of the 100 GBA cell lines, 50 were utilized for real-time PCR analysis to investigate the transcription levels, while the remaining 50 were used to assess the enzyme-specific activity of GCase variants by fluorimetric assay. Furthermore, we determine mRNA secondary structures of GBA variants using the ViennaRNA and three-dimensional structures of the GCase-Opt and GCase-7 enzymes were elucidated using the AlphaFold2. Results and discussion: Quantitative real time PCR revealed that for 4 GBA transcripts (GBA-opt, GBA-7, GBA-9 and GBA-12) under control of hEF1A promoter showed higher expression in 293FT cells compared with CMV promoter (p < 0.05). Among the GBA variants, we observed that 293FT_GBA-7 cells express 5.2-fold higher transcript levels compared with 293FT_GBA-opt cells (p < 0.05). In cells lines co-transfected with GFP and GBA, we observed that comparing normalized activity values while considering transfection efficiencies can provide a more accurate assessment of the enzyme catalytic properties. We also screened the cell lysates for GCase specific activity. We observed that 293FT_GBA-7 cells and 293FT_GBA-Opt cells produced 507.6 ± 38.16 and 426.6 ± 25.25 nmol substrate hydrolyzed/mg protein/h, respectively. For the other transgenic 293FT cell lines these values were lower. Also, for 293FT transfected with mock plasmid this value was 82.92 ± 5.83 nmol/mg/h, respectively (p = 0.014). The 3D structures of GCase-Opt and GCase-7 revealed a high degree of alignment with the Homo sapiens enzyme (UniProt P04062). Conclusions: These findings demonstrate the promise of using in silico molecular evolution and synthetic biology approaches for developing enhanced enzyme variants for Gaucher disease, and suggest that GBA-7 could be a viable candidate for further investigation for replacement therapy in GD. Additional studies are warranted to investigate the biodistribution and long-term effects of GBA-7 in animal models.

PDF file - 80K, ummary of correlation of linear regression calculations for UNG, SMUG1, TDG, MBD4, POLβ, TOPOIIα, and TYMS.

PDF file - 65K, Description of cell lines used in this study.

BACKGROUND:Premature aging has been identified as a global risk factor for cancer. Causes of premature aging are multifactorial, including inflammation, infection, chronic stress, and lifestyle factors. METHOD:We evaluated whether premature aging in people living with HIV (PLWH) was associated with antiretroviral therapy (ART) or the diagnosis of cancer. We used well-established DNA methylation patterns to assess premature aging, using Horvath et al., in individuals with HIV located in Cleveland, Ohio and compared these to standardized datasets of US historical blood samples. Some of the PLWH developed cancer over time. RESULTS:We found that DNA methylation analysis identified accelerated aging in PLWH whereas ART therapy mitigated the advancement of DNA methylation age. A variety of cancers were observed in this population, but a cancer diagnosis was not significantly associated with more advanced DNA methylation age. CONCLUSION:We find that the age acceleration detected in PLWH is mitigated by ART therapy and is not further accelerated by a diagnosis of cancer.

PDF file - 587K, Expression of selected pathway specific genes in pemetrexed-resistant sublines.

Supplementary Figure Legends

e24169 Background: Cannabis is gaining popularity worldwide including in the United States with 37 states legalizing access to cannabis. Limited data exist regarding why patients are using cannabis while receiving anticancer treatments. Methods: Patients with cancer who received treatment at a National Cancer Institute (NCI) designated CCC were approached from August of 2021 to August of 2022 to participate in a cross-sectional survey as part of a national NCI-funded effort. Consenting patients completed a survey during a medical visit asking about cannabis use (past and present), reason for use (including if cancer-related), type of cannabis, and frequency of use. A follow-up phone call occurred within 30 days to confirm medications used, and to collect further details about cannabis use, if reported. A healthcare provider survey was also conducted to explore perspectives regarding patients’ use of cannabis during active treatment. Results: A total of 315 cancer patients completed the survey (43% response rate) with a median age of 60.7 (±12.8), 58% female, and a variety of cancer types (hematologic 21.3%, breast 17%, gastrointestinal 11.7%, lung 10.2%). The respondents were of a diverse racial/ethnic backgrounds (White 61.6%, Black 22.5%, Other 7.3%), education (high school 27%, college 42.5%, post-graduate 14.9%), and employment status (retired 35.2%, employed 25.7%, unemployed or disabled 21.9%). Among respondents, nearly half (48.4%) had used cannabis before, one-third (32.1%) had used cannabis since their cancer diagnosis, and almost one in five (18.5%) were currently using cannabis. Among current or past users of cannabis the forms used were smoked (31%), gummy (29%), liquid (19%), edible (7%), and pill (6%). The most common reported reasons for use by respondents were insomnia (79%), pain (72%), mood (68%), poor appetite (68%), and gastrointestinal symptoms (51%). For the clinician survey, a total of 165 completed surveys (25% response rate) were received. Approximately half of respondents were nurses (54% nurses, 22% advance practice providers, and 21% physicians) and of White race (73%) with a mean age of 46 years. Approximately one-fourth (24%) reported discussing the topic of cannabis and about half of these conversations (45%) were initiated by the respondent. The healthcare providers estimated 10% of patients with cancer overall were using cannabis and that one in four patients (23%) were using cannabis during cancer treatment. Four in ten (39%) clinicians felt comfortable in providing guidance to patients about cannabis use, and only one in eight (13%) felt knowledgeable about the topic of cannabis. Conclusions: Approximately one-fifth of patients with cancer receiving treatment are actively using cannabis, despite limited data regarding the efficacy of cannabis. Management of cancer symptoms were among key reasons that cancer patients used cannabis.

Objective: To compare cytogenetic abnormalities among people living with HIV (PLWH) with and without previous exposure to Mycobacterium tuberculosis (Mtb) (both latent tuberculosis infection [LTBI] and active tuberculosis [TB]). Methods: Adult PLWH (& GE;18 years) were randomly selected at three HIV clinics in Uganda. Previous active TB was confirmed in the clinics' TB records. LTBI was defined as a positive QuantiFERON-TB Gold Plus assay. Participants' buccal mucosal exfoliated cells were examined (per 2000 cells) using the buccal micronucleus assay for chromosomal aberrations (micronuclei and/or nuclear buds), cytokinetic defects (binucleated cells), proliferative potential (normal differentiated cells and basal cell frequency) and/or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells). Results: Among 97 PLWH, 42 (43.3%) had exposure to Mtb;16 had previous successfully treated active TB and 26 had LTBI. PLWH with exposure to Mtb had a higher median number of normal differentiated cells (1806.5 [1757.0 - 1842.0] vs. 1784.0 [1732.0 - 1843.0], p = 0.031) and fewer karyorrhectic cells (12.0 [9.0 - 29.0] vs. 18.0 [11.0 - 30.0], p = 0.048) than those without. PLWH with LTBI had fewer karyorrhectic cells than those without (11.5 [8.0 - 29.0] vs. 18.0 [11 - 30], p = 0.006). Conclusion: We hypothesized that previous exposure to Mtb is associated with cytogenetic damage among PLWH. We found that exposure to Mtb is associated with more normal differentiated cells and less frequent karyorrhexis (a feature of apoptosis). It is unclear whether this increases the propensity for tumorigenesis.

Figure S2 shows UDG depletion by two other different shRNAs selectively sensitizes p53 KO cells to 5-FdU.