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Endemic Burkitt lymphoma (eBL) is a pediatric cancer coendemic with malaria in sub-Saharan Africa, suggesting an etiological link between them. However, previous cross-sectional studies of limited geographic areas have not found a convincing association. We used spatially detailed data from the Epidemiology of Burkitt Lymphoma in East African Children and Minors (EMBLEM) study to assess this relationship. EMBLEM is a case–control study of eBL from 2010 through 2016 in six regions of Kenya, Uganda, and Tanzania. To measure the intensity of exposure to the malaria parasite, Plasmodium falciparum , among children in these regions, we used high-resolution spatial data from the Malaria Atlas Project to estimate the annual number of P. falciparum infections from 2000 through 2016 for each of 49 districts within the study region. Cumulative P. falciparum exposure, calculated as the sum of annual infections by birth cohort, varied widely, with a median of 47 estimated infections per child by age 10, ranging from 4 to 315 infections. eBL incidence increased 39% for each 100 additional lifetime P. falciparum infections (95% CI: 6.10 to 81.04%) with the risk peaking among children aged 5 to 11 and declining thereafter. Alternative models using estimated annual P. falciparum infections 0 to 10 y before eBL onset were inconclusive, suggesting that eBL risk is a function of cumulative rather than recent cross-sectional exposure. Our findings provide population-level evidence that eBL is a phenotype related to heavy lifetime exposure to P. falciparum malaria and support emphasizing the link between malaria and eBL.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

As many as one third of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor deficiency in some DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DDR inhibitors. The concept of synthetic lethality has been successfully applied to develop strategies to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful particularly in Homologous Recombination Deficiency (HRD) cancers. Other agents have also been designed to target these DDR deficiencies in cancer cells and have potential to be clinically beneficial. LP-184 is a next generation acylfulvene prodrug metabolized to an active compound by prostaglandin reductase 1 (PTGR1) that is often elevated in multiple tumors. LP-184-induced DNA lesions are likely repaired by the Nucleotide Excision Repair (NER) and Homologous Repair (HR) pathways. Reduced activities of multiple DDR-related pathways in solid tumor models correlated with increased sensitivities to LP-184 as indicated by Gene Set Enrichment Analysis. Chinese Hamster Ovary cells with NER mutations (ERCC1/2/6) were at least 4 fold more sensitive to LP-184 relative to otherwise isogenic parental cells. ERCC2 or BRCA2 depletion enhanced LP-184 sensitivity 5-8X in a metastatic prostate cancer cell line. LP-184 showed ~120 fold higher potency (IC50 77 nM) compared to Olaparib in HRD prostate cancer organoid model LuCaP 96 (BRCA2/CHEK2 inactivating mutations). Across 14 patient-derived pancreatic, lung and prostate tumor models harboring HR mutations (BRCA1/2, CHEK1/2, ATM/ATR, PALB2, PARP1/2, RAD51, FANCA/B), LP-184 showed ex vivo IC50s in the range of 30-300 nM, whereas Olaparib IC50s ranged from 1700-6900 nM. LP-184 treatment resulted in complete tumor regression (107-141% TGI) in 10/10 HR deficient triple negative breast cancer PDX models of which 7/10 were resistant Olaparib/Niraparib and to Doxorubicin/Cyclophosphamide. LP-184 treatment resulted in complete and durable tumor regression in HRD (BRCA1/ATR mutant) pancreatic cancer PDX models (112-140% TGI). In the ovarian cancer cell line OVCAR3 having HRD/NERD phenotype, LP-184 IC50 was 13 nM, Olaparib IC50 was 145 nM and their combination Bliss Synergy Score was 16 indicating synergy. LP-184 is synthetic lethal resulting from its abilities to cause unresolvable DNA damage if tumor cells express high PTGR1 and are deficient in NER/HR genes. Unlike PARPi, LP-184 has striking activity in both NERD as well as HRD cancers. These strong data have prompted the development of a soon to be launched clinical trial to translate the broad preclinical anticancer activity of LP-184 in solid tumors with HR/NER pathway defects, such as pancreatic, prostate, ovarian and breast cancers. Citation Format: Aditya Kulkarni, Jianli Zhou, Neha Biyani, Kishor Bhatia, Panna Sharma. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6241.

Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to prolong survival of patients with newly diagnosed MGMT-methylated GBM. Blood-brain barrier (BBB) permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT unmethylated GBM) are desperately needed. Lantern Pharma is advancing LP-184, an acylfulvene prodrug activated by the enzyme PTGR1, in GBM. In an MGMT expressing TMZ resistant GBM PDX model, LP-184 was found to be ~5000X more potent than TMZ. DNA damage induced by acylfulvenes is primarily repaired via transcription-coupled nucleotide excision repair (TC-NER) mediated by ERCC complexes and gene set variation analyses associate LP-184 cell sensitivity with low TC-NER. ERCC3 is a key NER factor that is proteolytically degraded in response to the FDA approved BBB permeable diuretic, spironolactone (SP). After verifying that spironolactone induces rapid ERCC3 degradation by up to 95% in GBM cells, we validated that SP-induced ERCC3 loss led to substantial increases in LP-184 response in vitro and in subcutaneous tumor xenografts. Co-treatment of GBM cells with LP-184 and 5 or 25 µM SP resulted in a 3-6 fold decrease in LP-184 IC50s in M1123, Mayo39 and U87 cultures in vitro. Mice bearing pre-established subcutaneous U87 xenografts were treated with SP alone, LP-184 alone or their combination. Tumors recurred after initial regression in 5/5 animals treated with LP-184 alone whereas tumors showed durable complete regression without recurrence in 4/5 animals treated with LP-184 + SP. LP-184 is a promising chemotherapeutic with potential clinical translation in GBM patients. Our results indicate that LP-184 is MGMT-agnostic and effective in TMZ-resistant pre-clinical GBM models. Our findings also identify ERCC3-dependent TC-NER activity as a determinant of LP-184 synthetic lethality predicting that LP-184’s therapeutic potential will be significantly enhanced in patients with intrinsic or spironolactone-induced NER deficient tumors.

Temozolomide (TMZ) is currently the most effective standard-of-care chemotherapy based on its ability to prolong survival of patients with newly diagnosed MGMT-methylated GBM. Blood-brain barrier permeable agents effective against TMZ-resistant GBMs (i.e. recurrent GBM, MGMT unmethylated GBM) are desperately needed. Lantern Pharma is currently developing LP-184, a hydroxyurea methyl acylfulvene derivative of irofulven guided by its AI platform RADRⓇ analysis showing that tumor cell sensitivity to LP-184 correlates positively with genes commonly upregulated in GBM and either associated with TMZ resistance (e.g. MGMT, UGDH) or tumor promotion (e.g. EGFR, ANXA2). In an in vitro 3D model that mimics the human BBB, the apparent permeability for LP-184 was 1.53E-04 cm/s at 30 minutes, comparable to 1.72E-04 cm/s for TMZ under identical conditions. LP-184 predominantly alkylates DNA at 3’-adenine predicting insensitivity to MGMT expression. In vitro cell toxicity assays in a panel of GBM cell lines and neurospheres reveal an IC50 range of 30 - 400 nM with the MGMT unmethylated cell line LN-18 being among the most sensitive. A single cycle of LP-184 (4 mg/kg, i.v, every other day X 4 doses) induced rapid and near complete regression of subcutaneous U87 xenografts pre-established in immune-deficient mice and statistically significantly prolonged survival of mice bearing pre-established orthotopic U87 xenografts (p < 0.0001). LP-184-induced DNA damage is associated with synthetic lethality in tumor cell lines with decreased expression of Nucleotide Excision Repair (NER) genes. Furthermore, interrogation of clinical databases for expression of 112 NER genes revealed that 25 - 37% of clinical GBM clusters in a low NER expression subgroup, predicting high sensitivity of low NER GBM subsets to LP-184. These findings identify LP-184 as a promising new alkylating agent and support its further development for GBM therapy.

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS andEpstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV's

Biomarker-based therapeutic strategies associated with increased efficacy, manageable toxicity and extended disease-free survival are urgently needed in pancreatic cancer (PaCa). 15-20% of PaCa carry mutations in DNA repair pathways (BRCA1/BRCA2/PALB2/RAD51/ATR/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PaCa. LP-184 is an acylfulvene analog that is converted to an active alkylating agent in the strict dependency on the oxidoreductase, prostaglandin reductase 1 (PTGR1). LP-184 induced tumor cell kill is enhanced in the presence of DNA damage repair (DDR) defects. Mutation or expression driven TC-NER and/or HR deficiency is expected to predispose PaCa cells to increased sensitivity to LP-184. To test the idea of LP-184 activity in DNA repair-deficient tumors, we evaluated the anti-tumor effects of LP-184 in mouse subcutaneous PDX models harboring pathogenic frameshift mutations in ATR (CTG-1522 model) or BRCA1 (CTG-1643 model) representing HR deficiency in PaCa. Two cycles of LP-184 treatment in CTG-1522 resulted in 140% tumor growth inhibition (TGI), relative to 80% TGI with gemcitabine in this FOLFIRONOX non-responder. Similarly, complete and durable tumor regression with 112% TGI was observed in CTG-1643 with single agent LP-184. There was 10 indicates synergy) of 12.37 for LP-184 + gemcitabine in Capan-1, and 14.08, 16.47 and 15.52 for LP-184 + spironolactone in Capan-1 (BRCA2 loss), Hs766t (ATR mutant) and Panc03.27 cell lines respectively were achieved. Taking advantage of the transcriptional regulation underlying the NRF2 mediated anti-oxidant response pathway, we reasoned that induction of PTGR1 expression, a downstream NRF2 target gene, in irradiated PaCa tumors would thereafter poise them for LP-184 treatment. Indeed, we found that Panc03.27 xenograft tumors treated with 3 mg/kg i.p. LP-184 + 4 Gy RT once weekly for 3 weeks were significantly smaller than those receiving LP-184 alone or RT alone, thus capitalizing on the opportunity to increase PTGR1 expression (and linked LP-184 anti-tumor cytotoxicity) selectively in irradiated tumors. Increased PTGR1 expression has emerged as a promising biomarker for LP-184 cytotoxicity. Moreover, LP-184 can become synthetically lethal in multiple contexts when combined with genetic or pharmacologic DDR pathway aberrations, or with RT. Based on these properties, we anticipate that LP-184 will extend therapeutic opportunities to a large subset of PaCa patients. Citation Format: Aditya Kulkarni, Diana Restifo, Troy Dos Santos, Dusica Cvetkovic, Joseph McDermott, Kishor Bhatia, Panna Sharma, Charlie Ma, Igor Astsaturov. LP-184, a tumor site activated small molecule therapeutic, is synthetically lethal in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B033.

Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL). Approximately 40%-50% of MCL patients carry inactivating mutations in the gene ataxia telangiectasia mutated (ATM), a core component of the cell's DNA damage response system. MCL cells with mutations in ATM are known to have increased chromosomal imbalance, copy number loss, and hypersensitivity to DNA-damaging agents. LP-184, a small-molecule DNA-damaging agent, is known to be synthetically lethal in tumors with DNA-repair deficiencies, including tumors with ATM mutations. However, DNA damage by LP-184 is dependent on activation by the oxidoreductase prostaglandin reductase 1 (PTGR1), which is expressed at low levels in many hematological cancers. We synthesized LP-284, an enantiomer of LP-184, which retains synthetic lethality in cancers with impaired DNA damage repair pathway genes (e.g., ERCC1, ERCC2, ERCC3, ERCC6), but LP-284's activity is independent of PTGR1 expression. We demonstrated that LP-284 has enhanced tumoricidal activity in multiple hematological cancer cell lines using a B-cell NHL panel. The IC

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in sub-Saharan African countries, however, few epidemiologic studies have been undertaken and none attempted enrolling cases from multiple countries. We therefore conducted a population-based case-control study of eBL in children aged 0-15 years old in six regions in Northern Uganda, Northern Tanzania and Western Kenya, enrolling 862 suspected cases and 2,934 population controls (response rates 98.5-100%), and processing ~40,000 vials of samples using standardized protocols. Risk factor questionnaires were administered, and malaria period prevalence was measured using rapid diagnostic tests (RDTs). A total of 80.9% of the recruited cases were diagnosed as eBL; 61.4% confirmed by histology. Associations with eBL risk were computed using logistic regression models adjusted for relevant confounders. Associations common in at least two countries were emphasized. eBL risk was decreased with higher maternal income and paternal education and elevated with history of inpatient malaria treatment >12 months before enrollment. Reporting malaria-attributed fever up to 6 months before enrollment and malaria-RDT positivity at enrollment were associated with decreased eBL risk. Conversely, reporting exposure to mass malaria suppression programs (e.g., indoor residual insecticide) was associated with elevated risk. HIV seropositivity was associated with elevated eBL risk, but the relative impact was small. The study shows that it is feasible to conduct networked, multisite population-based studies of eBL in Africa. eBL was inversely associated with socioeconomic status, positively associated with inpatient malaria treatment 12 months ago and with living in areas targeted for malaria suppression, which support a role of malaria in eBL.

The burden of Plasmodium falciparum (Pf) malaria in Kenya is decreasing; however, it is still one of the top 10 causes of morbidity, particularly in regions of western Kenya. Between April 2015 and June 2016, we enrolled 965 apparently healthy children aged 0-15 years in former Nyanza and Western Provinces in Kenya to characterize the demographic, geographic, and household risk factors of asymptomatic malaria as part of an epidemiologic study to investigate the risk factors for endemic Burkitt lymphoma. The children were sampled using a stratified, multistage cluster sampling survey design. Malaria was assessed by rapid diagnostic test (RDT) and thick-film microscopy (TFM). Primary analyses of Pf malaria prevalence (pfPR) are based on RDT. Associations between weighted pfPR and potential risk factors were evaluated using logistic regression, accounting for the survey design. Plasmodium falciparum malaria prevalence was 36.0% (27.5%, 44.5%) by RDT and 22.3% (16.0%, 28.6%) by TFM. Plasmodium falciparum malaria prevalence was positively associated with living in the lake-endemic area (adjusted odds ratio [aOR] 3.46; 95% confidence interval [95% CI] 1.63, 7.37), paternal occupation as peasant farmer (aOR 1.87; 1.08, 3.26) or manual laborer (aOR 1.83; 1.00, 3.37), and keeping dogs (aOR 1.62; 0.98-2.69) or cows (aOR 1.52; 0.96-2.40) inside or near the household. Plasmodium falciparum malaria prevalence was inversely associated with indoor residual insecticide spraying (IRS) (aOR 0.44; 0.19, 1.01), having a household connected to electricity (aOR 0.47; 0.22, 0.98), and a household with two (aOR 0.45; 0.22, 0.93) or ≥ three rooms (aOR 0.41; 0.18, 0.93). We report high but geographically heterogeneous pfPR in children in western Kenya and significant associations with IRS and household-level socioeconomic factors.

Background Falciparum and endemic Burkitt lymphoma (eBL) are co-endemic in Africa, but the malaria experience in eBL patients is unknown. A lower prevalence of falciparum has been reported in eBL patients, but those results are anecdotally attributed to pre-enrollment anti-malaria treatment. Methods We studied 677 eBL patients and 2920 community controls aged 0–15 years enrolled in six regions in Uganda, Tanzania, and Kenya during 2010–2016. Falciparum was diagnosed using thick blood film microscopy (TFM) and antigen-capture rapid diagnostic tests (RDTs). Guardians of the children answered a 40-item structured questionnaire about their child’s pre-enrollment lifetime malaria history and treatment, demographics, socioeconomics, animal exposures, fevers, and hospitalizations. We utilized exploratory factor analysis to reduce the 40 questionnaire variables into six factors, including Inpatient malaria and Outpatient malaria factors that were surrogates of pre-enrollment anti-malaria treatment. The six factors accounted for 83–90% of the variance in the questionnaire data. We calculated odds ratios and 95% confidence intervals (OR 95% CI) of association of eBL with falciparum positivity, defined as positive both on TFM or RDTs, or only RDTs (indicative of recent infection) or TFM (indicative of current falciparum infection) versus no infection, using multivariable logistic regression, controlling for group of age (0–2, 3–5, 6–8, 9–11 and 12–15 years), sex, and study site and the afore-mentioned pre-enrollment factors. Results The prevalence of falciparum infection was 25.6% in the eBL cases and 45.7% in community controls (aOR = 0.43, 95% CI: 0.40, 0.47; P falciparum infection (6.9% versus 13.5%, aOR = 0.44, 95% CI: 0.38, 0.50; P falciparum infection (18.7% versus 32.1%, aOR = 0.47, 95% CI: 0.43, 0.51; P falciparum infection did not change when we adjusted for pre-enrollment factors (aORs = 0.46, =0.44, and = 0.51, respectively) were significantly lower in stratified analysis for any infection in children Conclusion Our study results reduce support for pre-enrollment antimalaria treatment as a sole explanation for the observed lower falciparum prevalence in eBL cases and open a space to consider alternative immunology-based hypotheses.

Background: Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in Africa and is linked to Plasmodium falciparum (Pf) malaria infection, one of the most common and deadly childhood infections in Africa, however, the role of Pf genetic diversity is unclear. A potential role of Pf genetic diversity in eBL has been suggested by a correlation of age-specific patterns of eBL with the complexity of Pf infection in Ghana, Uganda, and Tanzania, as well as a finding of significantly higher Pf genetic diversity, based on a sensitive molecular barcode assay, in eBL cases than matched controls in Malawi. We examined this hypothesis by measuring diversity in Pf-serine repeat antigen-5 (Pfsera5), an antigenic target of blood-stage immunity to malaria, among 200 eBL cases and 140 controls, all Pf polymerase chain reaction (PCR)-positive, in Malawi. Methods: We performed Pfsera5 PCR and sequencing (~3.3 kb over exons II–IV) to determine single or mixed PfSERA5 infection status. The patterns of Pfsera5 PCR positivity, mixed infection, sequence variants, and haplotypes among eBL cases, controls, and combined/pooled were analyzed using frequency tables. The association of mixed Pfsera5 infection with eBL was evaluated using logistic regression, controlling for age, sex, and previously measured Pf genetic diversity. Results: Pfsera5 PCR was positive in 108 eBL cases and 70 controls. Mixed PfSERA5 infection was detected in 41.7% of eBL cases versus 24.3% of controls, the odds ratio (OR) was 2.18, and the 95% confidence interval (CI) was 1.12–4.26, which remained significant in adjusted results (adjusted odds ratio [aOR] of 2.40, 95% CI of 1.11–5.17). A total of 29 nucleotide variations and 96 haplotypes were identified, but these were unrelated to eBL. Conclusions: Our results increase the evidence supporting the hypothesis that infection with mixed Pf infection is increased with eBL and suggest that measuring Pf genetic diversity may provide new insights into the role of Pf infection in eBL.

Background Non-targeted cytotoxics with anticancer activity are often developed through preclinical stages using response criteria observed in cell lines and xenografts. A panel of the NCI-60 cell lines is frequently the first line to define tumor types that are optimally responsive. Open data on the gene expression of the NCI-60 cell lines, provides a unique opportunity to add another dimension to the preclinical development of such drugs by interrogating correlations with gene expression patterns. Machine learning can be used to reduce the complexity of whole genome gene expression patterns to derive manageable signatures of response. Application of machine learning in early phases of preclinical development is likely to allow a better positioning and ultimate clinical success of molecules. LP-184 is a highly potent novel alkylating agent where the preclinical development is being guided by a dedicated machine learning-derived response signature. We show the feasibility and the accuracy of such a signature of response by accurately predicting the response to LP-184 validated using wet lab derived IC50s on a panel of cell lines. Results We applied our proprietary RADR® platform to an NCI-60 discovery dataset encompassing LP-184 IC50s and publicly available gene expression data. We used multiple feature selection layers followed by the XGBoost regression model and reduced the complexity of 20,000 gene expression values to generate a 16-gene signature leading to the identification of a set of predictive candidate biomarkers which form an LP-184 response gene signature. We further validated this signature and predicted response to an additional panel of cell lines. Considering fold change differences and correlation between actual and predicted LP-184 IC50 values as validation performance measures, we obtained 86% accuracy at four-fold cut-off, and a strong (r = 0.70) and significant (p value 1.36e−06) correlation between actual and predicted LP-184 sensitivity. In agreement with the perceived mechanism of action of LP-184, PTGR1 emerged as the top weighted gene. Conclusion Integration of a machine learning-derived signature of response with in vitro assessment of LP-184 efficacy facilitated the derivation of manageable yet robust biomarkers which can be used to predict drug sensitivity with high accuracy and clinical value.

More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC.

Additional file 1: Figure S1. Flow chart describing statistical methodology.

Additional file 2: Figure S2. Comparison of the different model accuracy on blind test data.

e14023 Background: Glioblastoma (GBM), a WHO Grade IV cancer, is the most common and most aggressive primary malignant brain tumor. Prognosis following best standard-of-care surgical resection, radiation and temozolomide (TMZ) chemotherapy is poor, with median overall survival 15-18 months. TMZ, which acts primarily by methylating DNA at O6-guanine, has marginal efficacy in ̃70% of patients due to tumor expression of O-6-methylguanine-DNA methyltransferase (MGMT) and there is no effective therapy at tumor recurrence. New treatment options are therefore desperately needed for GBM patients. LP-184 is an acylfulvene-derived prodrug that is activated by the oxidoreductase PTGR1 and primarily alkylates adenine at the N3-position. Bioinformatic analysis showed that PTGR1 is elevated in clinical GBM tumor samples compared to normal brain. LP-184 response is influenced by tumor DNA repair status, and acylfulvene-induced damage is mainly repaired by the transcription-coupled nucleotide-excision repair (TC-NER) pathway. Spironolactone, an FDA approved, blood-brain-barrier permeable diuretic, induces degradation of ERCC3 protein, a key TC-NER component. Response of NER-deficient GBM was tested through co-administration of LP-184 and spironolactone. Methods: Cell viability assays with exposure to LP-184, with and without spironolactone, were used to determine IC50 values in vitro. Subcutaneous or intracranial tumor xenograft models were used to determine tumor volume or survival differences between control and LP-184 treated mice. Results: Nanomolar concentrations of LP-184 inhibited in vitro viability of multiple GBM cell models including 4 NCI-60 GBM cell lines (mean IC50= 286 nM), the TMZ-resistant LN-18 cell line (IC50= 46 nM), M1123 GBM neurosphere line (IC50= 151 nM), and a low passage GBM neurosphere isolate (IC50= 306 nM). Co-treatment with spironolactone resulted in a 3-6 fold decrease in LP-184 IC50 values in M1123, Mayo39, and U87 cultures. Pharmacokinetic analyses in SCID mice following a single LP-184 infusion (4 mg/kg, i.v.) showed favorable CNS bioavailability with brain/plasma ratio 0.11 (Cmax= 839 nM) and brain tumor/plasma ratio 0.2 (Cmax= 2.53 µM). LP-184 (4mg/kg i.v., q.o.d X 4) induced complete regression of all pre-established s.c. U87 xenografts (N = 5) and 3 of 6 M1123 s.c. xenografts (mean tumor volumes: 1456 ± 349 mm 3 controls vs 84 ± 39 LP-184, p < 0.001) and statistically significantly prolonged survival of mice bearing pre-established orthotopic U87 and M1123 xenografts (p < 0.0001). Conclusions: These preclinical results establish LP-184 as a promising new agent for future clinical testing in patients with GBM and suggest that LP-184 may be particularly efficacious in patients with intrinsic or spironolactone-induced NER deficient tumors.

Background Endemic Burkitt lymphoma (eBL) is an aggressive B cell non-Hodgkin lymphoma associated with antigenic stimulation from Plasmodium falciparum malaria. Whether eBL risk is related to malaria parasite density is unknown. To address this issue, children with eBL, asymptomatic and clinical malaria, as a surrogate of malaria parasite density, were assessed. Methods Malaria-related laboratory results (parasite density, haemoglobin, platelet count, and white cell count [WBC]) count) were compiled for 4019 eBL cases and 80,532 subjects evaluated for asymptomatic malaria or clinical malaria (severe malaria anaemia, hyperparasitaemia, cerebral malaria, malaria prostration, moderate malaria, and mild malaria) in 21 representative studies published in Africa (mostly East Africa) and 850 eBL cases and 2878 controls with primary data from the Epidemiology of Burkitt Lymphoma in East African Children and Minors (EMBLEM) case–control study in Uganda, Tanzania, and Kenya. The average values of malaria-related laboratory results were computed by condition and trends across single-year age groups were assessed using regression and spline models. Results Overall, malaria infection or malaria was diagnosed in 37,089 of children compiled from the literature. Children with eBL and asymptomatic parasitaemia/antigenaemia, but not those with clinical malaria, were closest in their mean age (age 7.1–7.2 vs. 7.4–9.8 years), haemoglobin level (10.0–10.4 vs. 11.7–12.3 g/dL), malaria parasite density (2800 vs. 1827–7780 parasites/µL), platelet count (347,000–353,000 vs. 244,000–306,000 platelets/µL), and WBC count (8180–8890 vs. 7100–7410 cells/µL). Parasite density in these two groups peaked between four to five years, then decreased steadily thereafter; conversely, haemoglobin showed a corresponding increase with age. Children with clinical malaria were markedly different: all had an average age below 5 years, had dramatically elevated parasite density (13,905–869,000 parasites/µL) and dramatically decreased platelet count ( Conclusions eBL and asymptomatic parasitaemia/antigenaemia, but not clinical malaria, were the most similar conditions with respect to mean age and malaria-related laboratory results. These results suggest that children with asymptomatic parasitaemia/antigenaemia may be the population at risk of eBL.

Platelet counts are decreased in Plasmodium falciparum malaria, which is aetiologically linked with endemic Burkitt lymphoma (eBL). However, the pattern of platelet counts in eBL cases is unknown. We studied platelet counts in 582 eBL cases and 2 248 controls enrolled in a case-control study in Uganda, Tanzania and Kenya (2010-2016). Mean platelet counts in controls or eBL cases with or without malaria-infection in controls versus eBLcases were compared using Student's t-test. Odds ratios (ORs) and two-sided 95% confidence intervals (95% CIs) were estimated using multiple logistic regression, controlling for age, sex, haemoglobin and white blood cell counts. Platelets were decreased with malaria infection in the controls [263 vs. 339 × 109 platelets/l, P

Endemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma associated with Plasmodium falciparum (Pf) malaria and Epstein-Barr virus (EBV) infections. Variation in the Human Leukocyte Antigen (HLA) system is suspected to play a role, but assessments using less accurate serology-based HLA typing techniques in small studies yielded conflicting results. We studied 200 eBL cases and 400 controls aged 0-15 years enrolled in northern Uganda and typed by accurate high-resolution HLA sequencing methods. HLA results were analyzed at one- or two-field resolution. Odds ratios and 95% confidence intervals (aOR, 95% CI) for eBL risk associated with common HLA alleles versus alleles that were rare (

Biomarker-based chemotherapy with increased efficacy and prolonged disease-free survival are urgently needed in pancreatic cancer (PDAC). A 15-20% subset of PDAC tumors carry mutations in DNA repair pathway (BRCA1/BRCA2/PALB2/RAD51/ATM/FANCD2). Additionally, mutations in nucleotide excision repair (NER) genes (ERCC2/3/4/5/6) have been reported in ~5% of PDAC. LP184 is a novel synthetic small molecule acylfulvene analog. Using precise synthetic chemistry, we determined that only a negative enantiomer of LP184 is converted to an active alkylating agent in the strict dependency on oxidoreductase, prostaglandin reductase 1 (PTGR1). By computational analyses, we demonstrate a strong positive correlation of LP184 sensitivity with PTGR1 transcript levels (r=0.89, p Citation Format: Diana Restifo, Aditya Kulkarni, Caleb Schimke, Joseph McDermott, Umesh Kathad, Kishor Bhatia, Panna Sharma, Igor Astsaturov. LP184, a novel alkylating agent, is highly effective in pancreatic cancers with DNA damage repair defects [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-036.

LP-184, or (-)-hydroxyurea methylacylfulvene, is a potent DNA alkylating agent that effectively kills solid tumors. It belongs to the acylfulvene compound family known to induce DNA lesions repaired by the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. Here, we show that LP-284, the synthetic positive enantiomer of LP-184, exhibited the greatest and broadest hematologic cancer antiproliferative activities among the 6 acylfulvenes, including illudin S, illudin M, Irofulven (LP-100), the semisynthetic racemic LP-184, the synthetic negative enantiomer LP-184, and LP-284. The distinct pharmacological activities of LP-284 may be due to differences in metabolic activation, transport, or affinity to cellular macromolecules. To determine whether metabolic activation plays a role, we compared the correlation between the expression of Prostaglandin Reductase 1 (PTGR1), the NADPH-dependent oxidoreductase known to convert Irofulven into its active metabolite, and the IC50 of LP-184, Irofulven, and LP-284. We found that the expression level of PTGR1 is highly correlated with LP-184 (r=0.88, p=8.4e-20) and Irofulven (r=0.71, p=4.7e-10) sensitivity, but not with LP-284 (r=-0.01, p=0.93). We also found that the average expression level of PTGR1 is significantly lower in hematologic cancer cell lines (n=180) than in solid tumor cell lines (n=856), indicating the existence of an alternative LP-284 activator in hematologic cells. Next, we checked mutation status, RNA expression, protein expression, and DNA methylation of 489 oxidoreductases, but none of the enzymes was highly correlated with LP-284 activity. To further explore the potential clinical application of LP-284 in hematologic cancers, we conducted cell viability assays in 18 hematologic cancer cell lines and found that LP-284 exhibited nanomolar potency in acute lymphocytic leukemia (average IC50: 351 nM), chronic myeloid leukemia (average IC50: 360 nM), B-cell lymphoma (average IC50: 366 nM), and Multiple Myeloma (MM, IC50: 334 nM). We also investigated the therapeutic potential of LP-284 in combination with spironolactone in treating MM. Spironolactone, an FDA approved drug for hypertension, degrades one of the key TC-NER players ERCC3 in MM, which in turn makes cells more vulnerable to helix-distorting DNA lesions likely caused by LP-284. While Spironolactone alone didn't cause cytotoxicity to the MM cell line RPMI8226, it reduced LP-284 IC50 by 2.4 fold. Taken together, we have demonstrated the importance of stereochemistry in acylfulvene activity. LP-284, likely to be activated through a different route, is a unique and potent acylfulvene for hematologic cancers. Additionally, pharmacological inhibition of the TC-NER pathway greatly promoted LP-284 cytotoxicity. We hypothesize that LP-284 induces DNA lesions, which may be lethal to TC-NER deficient cells and may block transcription of short-lived fusion genes that are essential for cancer cell survival until repaired. Therefore, our discovery of the novel enantiomer LP-284 may provide a targeted therapy option for hematologic cancers with compromised DNA repair. Disclosures Zhou: Lantern Pharma: Current Employment. Biyani: Lantern Pharma: Current Employment. Kathad: Lantern Pharma: Current Employment, Current equity holder in publicly-traded company. Kulkarni: Lantern Pharma: Current Employment. McDermott: Lantern Pharma: Current Employment. Bhatia: Lantern Pharma: Current Employment.

Prostate cancer (CaP) remains the most commonly diagnosed malignancy and the second leading cause of cancer related deaths in men in the US. While the 5-year survival rate for patients with localized CaP is over 99%, it is only 30% for patients with distant metastases. Despite impressive advances in the treatment of metastatic CaP with more efficacious inhibitors along the androgen/androgen receptor axis, eventual development of incurable metastatic Castration Resistant Prostate Cancer (mCRPC) is inevitable, necessitating the development of newer therapeutic strategies. Comprehensive evaluations of CaP genomes from localized and metastatic CaP have revealed that subsets harboring mutations in certain DNA Damage Repair Genes (DDRGs) account for up to 30% of patients. LP184, a novel alkylating agent belonging to the acylfulvene (AF) class is currently in preclinical development, having demonstrated appreciable anticancer activity in multiple prostate tumor models. LP184 exhibits nanomolar potency (20 - 350 nM) against widely used CaP cell lines in 2D culture while having reduced cytotoxicity in a non-tumor prostate epithelial cell line. In CaP cell lines, LP184 turned out equipotent as standard chemotherapeutic Docetaxel, 100-2000 times more potent than another alkylating agent Cisplatin, and 100-9000 times more potent than PARP inhibitor, Olaparib. LP184 is also effective in 3 CaP organoid models 3D cultured from patient-derived xenografts. We hypothesize that LP184 exhibits potent inhibitory effects on CaP cells through a compromised DNA repair milieu of mCRPC due to damaging DDRG mutations in BRCA2, FANCI, ERCC3, ERCC6, FANCG, PALB2 and ATM represented by the in vitro CaP models tested. DNA damage resulting from AF treatment is known to be processed exclusively via transcription coupled nucleotide excision repair. Consistent with this, we observed enhanced LP-184 sensitivity in a cell line model with truncated ERCC3 relative to the otherwise syngeneic parental cell line. Based on Connectivity Map (CMAP) analysis of full transcriptome profiling data on LP184 treatment in selected CaP cell lines, we identified potential drug classes including PARP inhibitors, proteasome inhibitors, topoisomerase inhibitors and MTOR inhibitors that emerged in silico as synergistic with LP184. This approach utilized matching of genes differentially expressed upon LP184 exposure compared to 5000 other small molecules within CMAP. Advancement of LP184, potentially exploiting recurrent and actionable DDRG mutations, is anticipated to be translated into future clinical trials for mCRPC, a lethal CaP that is responsible for ~33,000 deaths/year in the US alone. Given that only a subset of patients responds to any single drug in this advanced CaP stage, LP184 stands out as a unique agent that may complement or synergize with drugs currently used in treatment of mCRPC and improve the outcomes for men with lethal CaP. Citation Format: Aditya Kulkarni, Partha Banerjee, Umesh Kathad, Kishor Bhatia, Panna Sharma. LP184, a novel alkylating agent, is efficacious in prostate cancer models with DNA damage repair defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1249.

Background Plasmodium falciparum (Pf) malaria infection is suspected to cause endemic Burkitt Lymphoma (eBL), but the evidence remains unsettled. An inverse relationship between sickle cell trait (SCT) and eBL, which supports that between malaria and eBL, has been reported before, but in small studies with low power. We investigated this hypothesis in children in a population-based study in northern Uganda using Mendelian Randomization. Methods Malaria-related polymorphisms (SCT, IL10, IL1A, CD36, SEMA3C, and IFNAR1) were genotyped in 202 eBL cases and 624 controls enrolled during 2010–2015. We modeled associations between genotypes and eBL or malaria using logistic regression. Findings SCT was associated with decreased risk of eBL (adjusted odds ratio [OR] 0·37, 95% CI 0·21–0·66; p = 0·0003). Decreased risk of eBL was associated with IL10 rs1800896-CT (OR 0·73, 95% CI 0·50–1·07) and -CC genotypes (OR 0·53, 95% CI 0·29–0·95, ptrend = 0·019); IL1A rs2856838-AG (OR 0·56, 95% CI 0·39–0·81) and -AA genotype (OR 0·50, 95% CI 0·28–1·01, ptrend = 0·0016); and SEMA3C rs4461841-CT or -CC genotypes (OR 0·57, 95% CI 0·35–0·93, p = 0·0193). SCT and IL10 rs1800896, IL1A rs2856838, but not SEMA3C rs4461841, polymorphisms were associated with decreased risk of malaria in the controls. Interpretation Our results support a causal effect of malaria infection on eBL., Highlights • Mendelian randomization analysis was done to assess a causal relationship between malaria infection and endemic Burkitt lymphoma in Uganda • Carriage of the sickle cell trait was associated with decreased risk of endemic Burkitt lymphoma • Heterozygous or homozygous minor alleles of IL10 rs1800896, IL1A rs2856838, and SEMA3C rs4461841 were associated with decreased risk of endemic Burkitt lymphoma • The inverse association between sickle cell trait and endemic Burkitt lymphoma supports a causal role of malaria in endemic Burkitt lymphoma

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy (IGH) and light chain (IGK/IGL) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK/IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK/IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Background: The discovery of Epstein–Barr virus (EBV) in Burkitt lymphoma tumors represented the first link between a virus and cancer in humans, but the underlying role of this virus in endemic Burkitt lymphoma remains unclear. Nearly all children in Burkitt lymphoma–endemic areas are seropositive for EBV, but only a small percentage develop disease. Variation in EBV-directed immunity could be an explanatory cofactor. Methods: We examined serum from 150 Burkitt lymphoma cases and 150 controls using a protein microarray that measured IgG and IgA antibodies against 202 sequences across the entire EBV proteome. Variation in the EBV-directed antibody repertoire between Burkitt lymphoma cases and controls was assessed using unpaired t tests. ORs quantifying the association between anti-EBV IgG response tertiles and Burkitt lymphoma status were adjusted for age, sex, and study year. Results: Thirty-three anti-EBV IgG responses were elevated in Burkitt lymphoma cases compared with controls (P ≤ 0.0003). Burkitt lymphoma–associated IgG elevations were strongest for EBV proteins involved in viral replication and antiapoptotic signaling. Specifically, we observed ORs ≥4 for BMRF1 (early antigen), BBLF1 (tegument protein), BHRF1 (Bcl-2 homolog), BZLF1 (Zebra), BILF2 (glycoprotein), BLRF2 [viral capsid antigen (VCA)p23], BDLF4, and BFRF3 (VCAp18). Adjustment for malaria exposure and inheritance of the sickle cell variant did not alter associations. Conclusions: Our data suggest that the anti-EBV serologic profile in patients with Burkitt lymphoma is altered, with strong elevations in 33 of the measured anti-EBV IgG antibodies relative to disease-free children. Impact: The Burkitt lymphoma–specific signature included EBV-based markers relevant for viral replication and antiapoptotic activity, providing clues for future Burkitt lymphoma pathogenesis research.

Populations in sub-Saharan Africa have historically been exposed to intense selection from chronic infection with falciparum malaria. Interestingly, populations with the highest malaria intensity can be identified by the increased occurrence of endemic Burkitt Lymphoma (eBL), a pediatric cancer that affects populations with intense malaria exposure, in the so called “eBL belt” in sub-Saharan Africa. However, the effects of intense malaria exposure and sub-Saharan populations’ genetic histories remain poorly explored. To determine if historical migrations and intense malaria exposure have shaped the genetic composition of the eBL belt populations, we genotyped ~4.3 million SNPs in 1,708 individuals from Ghana and Northern Uganda, located on opposite sides of eBL belt and with ≥ 7 months/year of intense malaria exposure and published evidence of high incidence of BL. Among 35 Ghanaian tribes, we showed a predominantly West-Central African ancestry and genomic footprints of gene flow from Gambian and East African populations. In Uganda, the North West population showed a predominantly Nilotic ancestry, and the North Central population was a mixture of Nilotic and Southern Bantu ancestry, while the Southwest Ugandan population showed a predominant Southern Bantu ancestry. Our results support the hypothesis of diverse ancestral origins of the Ugandan, Kenyan and Tanzanian Great Lakes African populations, reflecting a confluence of Nilotic, Cushitic and Bantu migrations in the last 3000 years. Natural selection analyses suggest, for the first time, a strong positive selection signal in the ATP2B4 gene (rs10900588) in Northern Ugandan populations. These findings provide important baseline genomic data to facilitate disease association studies, including of eBL, in eBL belt populations., Author summary We present a genome-wide analyses of genetic structure, gene flow, and natural selection in Ghana and Northern Uganda populations, both residing in the Sub-Saharan eBL belt, a region with intense falciparum malaria transmission and high endemic Burkitt Lymphoma (eBL) incidence. These populations are from different ethnolinguistic groups and are located 2400 miles apart in sub-Saharan Africa. We characterized genetic composition of these populations in the context of 22 additional African populations and present evidence for gene flow events that occurred in the last 3000 years, possibly related to regional migrations in Western Africa and major migrations involving Nilotic, Cushitic, and Bantu groups. We identified in Northern Ugandans a strong signal of malaria-driven selection in the ATP2B4 gene coding for a calcium transporter expressed in erythrocytes. Characterization of biological relationships between the ATP2B4 gene and malaria may inform the investigation of complex genomic disease associations in eBL belt populations.

Background: Endemic Burkitt lymphoma (eBL) is an aggressive childhood B-cell lymphoma linked to Plasmodium falciparum (Pf) malaria in sub-Saharan Africa. We investigated antibody reactivity to several human receptor-binding domains of the Pf erythrocyte membrane protein 1 (PfEMP1) that play a key role in malaria pathogenesis and are targets of acquired immunity to malaria. Methods: Serum/plasma IgG antibody reactivity was measured to 22 Pf antigens, including 18 to PfEMP1 CIDR domains between cases and controls from two populations (149 eBL cases and 150 controls from Ghana and 194 eBL cases and 600 controls from Uganda). Adjusted odds ratios (aORs) for case-control associations were estimated by logistic regression. Findings: There was stronger reactivity to the severe malaria associated CIDRα1 domains than other CIDR domains both in cases and controls. eBL cases reacted to fewer antigens than controls (Ghana: p = 0·001; Uganda: p = 0·03), with statistically significant lower ORs associated with reactivity to 13+ antigens in Ghana (aOR 0·39, 95% CI 0·24–0·63; pheterogeneity = 0·00011) and Uganda (aOR 0·60, 95% CI 0.41–0·88; pheterogeneity = 0·008). eBL was inversely associated with reactivity, coded as quartiles, to group A variant CIDRδ1 (ptrend = 0·035) in Ghana and group B CD36-binding variants CIDRα2·2 (ptrend = 0·006) and CIDRα2·4 (ptrend = 0·033) in Uganda, and positively associated with reactivity to SERA5 in Ghana (ptrend = 0·017) and Uganda (ptrend = 0·007) and group A CIDRα1·5 variant in Uganda only (ptrend = 0·034). Interpretation: eBL cases reacted to fewer antigens than controls using samples from two populations, Ghana and Uganda. Attenuated humoral immunity to Pf EMP1 may contribute to susceptibility to low-grade malaria and eBL risk. Funding: Intramural Research Program, National Cancer Institute and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services.

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed < 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

A novel clinical agent, LP-184 is being developed in conjunction with a dedicated machine learning-guided response signature, to allow optimal benefit of LP-184 through genomics-guided therapy. To define correlates of tumor genomics with sensitivity to LP-184, we used RADR™ (Response Algorithm for Drug Positioning and Rescue), a proprietary artificial intelligence (AI)-driven platform, and CellMinerCDB (cross database)™, a systems biology platform integrating molecular and pharmacological datasets on cancer cell lines. The input for our correlational analyses, include LP-184 IC50 data on NCI-60 cell line panel representing drug response, and multi-omics data on these cell lines. CellMinerCDB™ based analysis of LP-184 using a Lasso regression model generated a 38 gene response signature that included expression of APP, NEK6, EGFR, SQSTM1, SLC25A42, SLC16A10, POLD1, SMARCC1, POLG2, CHEK1. In building the model, the signature demonstrates a sensitivity R2 = 0.98 between observed and predicted IC50 values. Additional omics data including methylation and protein levels validate the relevance of several signature genes: APP, NEK6, EGFR, SQSTM1. Concurrently, a RADR™ based analysis of LP-184 using an Artificial Neural Network (ANN) classifier model generated a 10 gene response signature with PTGR1 and PTPN14 as the top weighted genes by relative importance. To establish a signature with highest possible sensitivity, we integrated these 2 additional genes in the 38 gene signature, creating a modified 40 gene signature now having an R2 = 0.99. We next used laboratory experimental studies to measure the performance of this signature. Using an independent set of 18 cell lines not used in signature development, we obtained IC50 values and categorized cell lines as sensitive or resistant. Our in silico results matched in vitro experimental results for 13/18 cell lines (72 percent accuracy). We further applied this 40 gene signature to interrogate 1036 cell lines representing a spectrum of tumor types in the Cancer Cell Line Encyclopedia (CCLE) and obtain a signature-derived IC50. The predicted IC50 ranges from 2.7nM to 114.6uM. The least responsive tumors represent hematological cancers, reproducing the observations made in the NCI-60 panel. 92 of 116 (79%) hematologic cancers in the CCLE database showed a predicted IC50 above 1µM with a median of 3.4uM. The top 279 cell line records with a predicted IC50 below 100nM represented solid tumors with NSCLC, renal, CNS and head & neck cancers as the most sensitive. In conclusion our results demonstrate that the development of LP-184 guided by tumor gene expression patterns modeled using a combination of algorithms and signatures provides a valuable component to the armamentarium of drugs in diverse solid tumors. Citation Format: Umesh Kathad, Aditya Kulkarni, Jean Philippe Richard, Terri Lehman, Rama Modali, Kishor Bhatia, Panna Sharma, Fathi Elloumi, Yves Pommier, William C. Reinhold. Machine learning-derived gene signature predicts strong sensitivity of several solid tumors to the alkylating agent LP-184 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2090.

e21660 Background: Lung cancer in non-smokers (LCINS) is the seventh leading cause of death among solid tumors. LCINS is more frequent in women, and the histological incidence of adenocarcinoma is higher among non-smokers. The mutation pattern by smoking status in non-small cell lung cancer (NSCLC) is distinct with genomic alterations in EGFR, MET and ROS1 being more frequent in non-smokers. Non-smoking status is the strongest clinical predictor of benefit from the EGFR TKIs. Lantern Pharma is developing LP-300/Dimesna, a non-toxic, well-tolerated, water-soluble disulfide as a combination therapy agent potentially indicated in female never-smokers with adenocarcinoma. Retrospective subgroup analyses of prior phase 3 trial data showed substantial overall survival benefit for LP-300 in combination with standard chemotherapy in patients with advanced NSCLC, especially in the female non-smoker adenocarcinoma subgroup (13.4 months in control arm to 27 months in treatment arm). Purpose of this work conducted at BioNumerik Pharmaceuticals was to determine if LP-300 modulates activity of selected receptor tyrosine kinases relevant to non-smoker NSCLC. Methods: In vitro luminescence-based kinase assays were used to evaluate the effect of LP-300 and LP-300-derived mesna-disulfide heteroconjugates, Erlotinib, and Crizotinib, in combination and as single agents, on EGFR, MET, and ROS1 kinase activity. Results: LP-300 inhibited WT EGFR kinase and MET kinase. LP-300 had a time-dependent inhibitory effect on ROS1 kinase activity. LP-300 and LP-300-derived mesna-disulfide heteroconjugates stimulated Erlotinib-mediated inhibition of WT and T790M EGFR. T790M EGFR is normally insensitive to Erlotinib but when LP-300 was used with Erlotinib, inhibition was more prominent. LP-300 also stimulated Crizotinib-mediated inhibition of MET kinase and displayed time-dependent stimulation of Crizotinib-mediated inhibition of ROS1 kinase. Conclusions: LP-300 is a multi-targeted modulator of enzymatic activities of EGFR, MET and ROS1, and potentiates the activity of Erlotinib and Crizotinib. EGFR family members contain conserved extracellular structures that are stabilized by disulfide bonds. Compounds that disrupt extracellular disulfide bonds could inactivate EGFR and potentially downregulate EGFR-dependent proliferative signaling in cancer cells. We propose LP-300 as a Disulfide Bond Disrupting Agent (DDA) that may complement existing EGFR-targeted agents functioning through alternate mechanisms.