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Background: Fostroxacitabine bralpamide (fostrox) is an orally administered liver-targeted troxacitabine-based nucleotide prodrug currently undergoing phase 1/2a clinical trial in advanced hepatocellular carcinoma (HCC), in combination with pembrolizumab or lenvatinib (NCT03781934). In phase 1 monotherapy fostrox has demonstrated proof-of-concept in advanced HCC, intrahepatic cholangiocarcinoma and liver metastasis from gastrointestinal solid tumors. Liver-selective fostrox-induced DNA-damage and tumor cell killing has the potential to enhance the anti-tumor activity in combination with checkpoint blockade, and inhibition of angiogenesis leads to increased levels of the active metabolite of fostrox. We therefore investigated a triple combination of fostrox with anti-PD1 and lenvatinib in nonclinical tumor models in vivo. Methods: Combinations of fostrox with anti-PD1 and lenvatinib treatment were evaluated in the subcutaneous syngeneic mouse CT26 model1. Groups of 8 mice were randomized using the Matched Distribution method based on tumor size day 1. Treatment was with fostrox (BID days 1-5, p.o.), anti-PD1 (BIW for 3 weeks, i.p.), and lenvatinib (QD for 21 days, p.o.). Tumours were measured three times weekly during the dosing phase, and statistical differences between the treatment groups was analyzed by two-way ANOVA. Pharmacodynamic response to fostrox, induction of DNA-damage (phospo-ser139-H2AX), was assessed by immunohistochemistry (IHC). Tumor infiltrating lymphocytes (TILs) were assessed by IHC evaluating the expression level of CD8, CD4, LAG-3, and PD-L1. Results: Treatment with the triple combination of fostrox (30mg/kg) plus anti-PD1 (3mg/kg) and lenvatinib (5mg/kg) showed a significantly (p Conclusions: The triple combination of fostrox with anti-PD1 and lenvatinib showed enhanced efficacy in a nonclinical tumor model, and changes in TILs were consistent with increased immune-mediated anti-tumor activity. The results indicate a potential for increased anti-tumor efficacy using a triple combination of fostrox plus checkpoint inhibition and anti-angiogenic therapy. 1The study was approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio UK, and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) Citation Format: Fredrik G. Oberg, Sujata Bhoi, Malene Jensen, Karin Tunblad, Hans Wallberg. A triple combination of fostrox (MIV-818) with immune checkpoint and kinase inhibition shows increased anti-tumor efficacy in vivo [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 2691.

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness. What's new? In chronic lymphocytic leukemia (CLL), cases with unmutated immunoglobulin receptors (U-CLL) are generally associated with inferior outcome, albeit still displaying considerable heterogeneity. Might such differences in CLL progression be explained by epigenetics? In this study, the authors found that an unusually aggressive subset of CLLs called subset #8 has a distinctive DNA-methylation profile. They also found that p63 is a novel pro-survival factor for CLL cells. These molecular studies may lead to new prognostic biomarkers, and possibly new therapeutic targets, for CLL.

Downregulation of miR26A1 has been reported in various B-cell malignancies; however, the mechanism behind its deregulation remains largely unknown. We investigated miR26A1 methylation and expression levels in a well-characterized series of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). From 450K methylation arrays, we first observed miR26A1 (cg26054057) as uniformly hypermethylated in MCL (n = 24) (all >75%), while CLL (n = 18) showed differential methylation between prognostic subgroups. Extended analysis using pyrosequencing confirmed our findings and real-time quantitative PCR verified low miR26A1 expression in both CLL (n = 70) and MCL (n = 38) compared to normal B-cells. Notably, the level of miR26A1 methylation predicted outcome in CLL, with higher levels seen in poor-prognostic, IGHV-unmutated CLL. Since EZH2 was recently reported as a target for miR26A1, we analyzed the expression levels of both miR26A1 and EZH2 in primary CLL samples and observed an inverse correlation. By overexpression of miR26A1 in CLL and MCL cell lines, reduced EZH2 protein levels were observed using both Western blot and flow cytometry. In contrast, methyl-inhibitor treatment led to upregulated miR26A1 expression with a parallel decrease of EZH2 expression. Finally, increased levels of apoptosis were observed in miR26A1-overexpressing cell lines, further underscoring the functional relevance of miR26A1. In summary, we propose that epigenetic silencing of miR26A1 is required for the maintenance of increased levels of EZH2, which in turn translate into a worse outcome, as shown in CLL, highlighting miR26A1 as a tumor suppressor miRNA.