This table shows purity of Subcellular Fractions.

This table shows IC50 values for antiproliferative activities toward EOC versus normal ovary cell lines by 5-substituted pyrrolo[3,2-d]pyrimidine inhibitors.

This table shows primer sequences for quantitative real-time RT-PCR.

This table shows information of ovarian cancer tissue cDNA array for real-time RT-PCR.

This figure shows generation of SKOV3 SHMT1 and SHMT2 knockdown (KD) cell lines.

This figure shows results of apoptosis assays with SKOV3 cells treated with AGF347.

This figure shows immunohistochemical staining of tumor microarray (TMA) for SHMT2 protein.

This figure shows purine nucleotides in SKOV3 EOC cells treated with pyrrolo[3,2-d]pyrimidine antifolates.

Menin scaffolds the oncogenic histone-lysine-N-methyltransferase (KMT2A)-fusion protein (FP) complex in KMT2A-r and wild-type KMT2A complex in NPM1-m acute myeloid leukemia (AML). Menin inhibitors (MIs) are effective in KMT2A-r AML and NPM1-m AML. However, not all patients respond to MIs as monotherapy. In this preclinical study, we demonstrate that the MI ziftomenib, in combination with the XPO1 inhibitor selinexor, synergistically inhibited the growth of multiple KMT2A-r and NPM1-m AML cell lines (CI<1). The combination suppressed colony formation in primary CD34+ KMT2A-r progenitor cells without affecting normal stem cells. Robust apoptosis and decreased G2/M populations were also evident. The combination downregulated HOXA9 and MEIS1 while upregulating monocytic differentiation marker CD11b in both the AML molecular signatures. RNA sequencing and proteomic analysis in KMT2A-r revealed suppression of multiple bona fide menin-KMT2A target genes. Our mechanistic studies also identified a novel role of XPO1 in stabilizing menin's binding to chromatin and its interactions with KMT2A and KMT2A/MLLT3. XPO1 inhibitor-mediated disruption of these interactions, particularly in combination with ziftomenib, synergistically impairs oncogenic transcriptional programs. In vivo, combination therapy improved survival in both MV4;11 and OCI-AML3 cell line and primary patient-derived KMT2A-r and NPM1-m AML xenograft models in NSG mice, effective even at reduced drug doses. These preclinical findings demonstrate that simultaneous inhibition of the menin-KMT2A interaction and XPO1 can be a more effective translational strategy for treating KMT2A-r and NPM1-m AML than MI monotherapy to deepen responses and delay/prevent relapses.

Myeloid leukemia associated with Down syndrome (ML-DS), as classified by WHO 2016, includes acute myeloid leukemia (AML) and myelodysplasia in children with DS. While ML-DS patients show high sensitivity to cytarabine (Ara-C)-based chemotherapy with better overall survival than non-DS AML patients, relapsed/refractory cases have dismal outcomes. This underscores the need to understand Ara-C-resistance mechanisms and develop effective therapies. The chromosome 21 gene, cystathionine-β-synthase (CBS), is significantly overexpressed in ML-DS cells. Overexpression of CBS leads to increased hydrogen sulfide (H2S) production, which reduces complex IV activity and oxidative phosphorylation (OXPHOS). OXPHOS has been shown to play an important role in Ara-C resistance in non-DS AML. Thus, in this study, we investigated the role of CBS as a regulator of OXPHOS and Ara-C response. We found that Ara-C-resistant ML-DS cells have lower CBS activity. Overexpression of CBS in an Ara-C-resistant ML-DS cell line resulted in increased H2S and Ara-C sensitivity and decreased both complex IV activity and OXPHOS. Knockdown of CBS in an Ara-C-sensitive ML-DS cell line increased OXPHOS and Ara-C resistance. However, complex IV activity decreased and H2S production was unchanged, indicating that CBS regulates OXPHOS through both a H2S-dependent and -independent mechanism. We further demonstrate that targeting both OXPHOS, using ONC213, and apoptosis, using venetoclax, results in synergistic induction of cell death in Ara-C-resistant ML-DS cells. This study identifies CBS as a regulator of OXPHOS and Ara-C response, while the combination of ONC213 and venetoclax offers a promising therapeutic approach for relapsed/refractory ML-DS, addressing key vulnerabilities to improve patient outcomes.

Background: Acute erythroid leukemia (AEL) or AML-M6 predominantly affects older adults and is rare in childhood. Compared with other AML subtypes, AEL remains relatively understudied because of its rarity. We established LS-CHM, a novel AEL cell line derived from the ascitic fluid of a patient with congenital leukemia. Interestingly, leukemic cells persisted in the ascitic fluid even after successful eradication from the bone marrow and extramedullary sites. Method: Leukemia cells from the ascites fluid exhibited robust proliferation in culture independent of cytokine requirement and were further characterized by flow cytometric immunophenotyping, cytogenetics, cell cycle and doubling time analysis, colony formation, genome and RNA sequencing, myeloid gene next generation sequencing, and cytotoxicity analysis. Results: LS-CHM displayed CD36, partial CD235a, CD31, CD43, and CD71 expression and demonstrated in vitro robust growth and high sensitivity to chemotherapeutic agents. A PDX mouse model showed development of leukemia. Genomic analysis revealed a frameshift BCOR mutation in the absence of additional mutations and downregulated TP53 expression with an exonic non-deleterious mutation. RNA sequencing of LS-CHM cells revealed upregulation of two cohesin complex genes, RAD21 and SMC3, whose high levels are associated with hematopoietic stem cell differentiation into erythroid lineage. Conclusions: LS-CHM represents the first congenital AEL-derived cell line, in contrast to the predominantly adult-origin and often secondary erythroid leukemia cell lines available currently. Thus, LS-CHM provides a unique pediatric and extramedullary AEL model, expanding the existing spectrum of AEL cell lines and offering valuable opportunities for biologic and therapeutic investigations.

This table shows information of epithelial ovarian cancer tissue microarray (TMA) for immunohistochemistry.

This table shows Kis for inhibition of one-carbon enzymes for 5-substituted pyrrolo[3,2- d]pyrimidine antifolates.

This figure shows purification of the recombinant human thymidylate synthase (TS) and in vitro inhibition by pyrrolo[3,2-d]pyrimidine antifolates.

Androgen receptor (AR)-dependent prostate cancer (PCa) cells require co-activation of ELK1 by AR to activate a critical set of cell cycle and mitosis genes, regardless of hormone - sensitivity. A small molecule antagonist (KCI807) that inhibits AR-dependent growth by selectively binding to AR and blocking its association with ELK1 is limited as a drug by auto-induced metabolism. Using structure-activity data, consistent with modeling a physically mapped KCI807 binding pocket, we developed a new class of compounds with a different core structure comprising 5-Hydroxy-2-(3-hydroxyphenyl)-1-methylquinolin-4(1H)-one (KCI830), with variable N- substituents. The compound with a N-2,2,2-trifluoroethyl substitution (KCI838) was the fastest acting and most potent inhibitor of AR-dependent cell growth and colony formation in PCa model cells, including exclusively AR splice variant-dependent and other enzalutamide-resistant cells, without affecting growth of AR-negative cell lines. Critical tests were conducted to establish that KCI838 recapitulates the previously elucidated mode of action of KCI807. KCI838 selectively inhibited ELK1-dependent vs. androgen response element (ARE)-driven promoter and gene activation by AR. KCI838 blocked AR binding to ELK1 in situ tested by BRET assay. Increasing the total cellular AR by ∼2-fold using ectopic AR expression caused the predicted change in drug dose-response profile for growth, implicating AR as the exclusive target for the activity of KCI838. KCI838's molecular scaffold conferred reduced enzyme induction in primary human hepatocytes and weakened interactions with human UGT1A1 and CYP1A2. In mice bearing an aggressive, enzalutamide-resistant patient-derived PCa tumor xenograft characteristically overexpressing prostatic acid phosphatase, daily bolus injections of a soluble 3'phosphate monoester prodrug of KCI838 (KCI838PME) progressively inhibited tumor growth, concomitant with tumor accumulation of active hydrophobic drug, without significant toxicity. Additionally, ALZET osmotic pumps were used to establish proof-of-concept for reversible in vivo anti-tumor activity of KCI838PME administered in a low dose, controlled release mode. The results warrant investigation of KCI838PME in a controlled-release formulation, to treat PCa that is resistant to current AR-targeted therapies while obviating the need for testosterone suppression.

This figure shows FRα, PCFT and RFC expression in EOC cell line models and EOC patient cDNAs.

Acute myeloid leukemia (AML) is an aggressive hematologic disease with a dismal prognosis. Each year in the US, there are approximately 20,000 new cases of AML and about 11,000 deaths. For adults, the 5-year overall survival rate is only 31.9%. One-third of AML patients harbor activating mutations in FMS-like tyrosine kinase 3 (FLT3) gene resulting in constitutive activation of downstream survival pathways. The most common FLT3 mutation is internal tandem duplication (FLT3-ITD), which occurs in approximately 25% of all AML patients. FLT3-ITD AML patients have reduced survival rates and an increased risk of relapse compared to AML patients with wild-type FLT3. Gilteritinib was recently approved for relapsed/refractory (R/R) FLT3-mutated AML patients. Although gilteritinib significantly improves clinical outcome for R/R FLT3-mutated AML patients, the 1-year survival rate is merely 37.1%, highlighting the need for new strategies for treating R/R FLT3-ITD positive AML. One common mechanism of resistance to FLT3 inhibition is inherent or acquired FLT3 mutations. To circumvent this, an alternative approach is to induce degradation of FLT3-ITD oncoprotein. Recently, the ubiquitin-specific peptidase 10 (USP10) was identified as a deubiquitinase (DUB) that stabilizes the FLT3-ITD oncoprotein, while sparing wild-type FLT3. The USP10 inhibitor Wu-5 downregulates FLT3-ITD and has antileukemic activity. Additionally, the USP7 inhibitor P22077 has demonstrated antileukemic activity and also targets USP10 in AML cells. While these covalent USP10 inhibitors are promising, they lack specificity for USP10 and/or potency. Therefore, there is an unmet need to develop potent USP10-selective inhibitors. We found that AML cells with acquired resistance to AraC (a main drug used for the treatment of AML) expressed high levels of FLT3-ITD and USP10 proteins, suggesting that degrading FLT3-ITD will show antileukemic activity against R/R AML cells. We used structure-activity relationship (SAR) and computational studies to develop non-covalent USP10 inhibitors. Our initial in vitro screening identified two compounds with antileukemic activity against FLT3-ITD AML cells with acquired resistance to AraC. Focusing on one of these novel USP10 inhibitors, GL-320, we show that it has significantly greater in vitro potency against AML cells than the reported USP10 inhibitors P-22077 and Wu-5. Cellular thermal shift assays show that GL-320 binds to USP10. Furthermore, GL-320 induces apoptosis in FLT3-ITD AML cells through inhibition of USP10 and subsequent downregulation of FLT3-ITD and PARP1 independent of caspase activation, and pharmacological inhibition of these two proteins induced apoptosis in a cooperative manner. Our results demonstrate that GL-320 is a non-covalent USP10 inhibitor that shows potent in vitro antileukemic activity against FLT3-ITD AML. Amirreza Samarbakhsh, Jianlei Zhao, Sadaf Dorandish, Jenna Thibodeau, Elyas Khan, Q. Ping Dou, Lisa Polin, Juiwanna Kushner, Jeffrey W. Taub, Yubin Ge, Navnath S. Gavande. Targeting oncogenic FLT3-ITD by USP10 inhibitors for the treatment of FLT3-ITD AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB426.

TNBC chemotherapeutics activate proinflammatory and oncogenic NF-κB pathways that involve CARP-1 interactions with NEMO, RIPK1, and STAT3 [Venkatesh JA et al. JBC 295 (11): 3532-3552, 2020; Venkatesh JA et al. Front. Oncol. 14:1376666, 2024]. To further investigate tumor growth, survival and metastasis potential of CARP-1, we generated multiple, human and murine cancer cells with homozygous CARP-1 knock-out (KO) by CRISPR-Cas9 technology. We next conducted high throughput proteomics analyses to identify proteins that were significantly up- or down-regulated in the CARP-1 KO cell lines relative to their CARP-1 expressing counterparts. We noted a consistent, and statistically significant reduction in levels of eukaryotic translation initiation factor (EIF4A)2 and HNRNPD (aka AU-rich RNA binding factor 1; AUF1), while Nestin levels were elevated in all CARP-1 KO cells. Our co-IP and western blot analyses revealed CARP-1 interactions with eIF4A and AUF1. EIF4A, the catalytic subunit of the eIF complex, functions to promote translation of different oncogenic, invasion, and metastasis-inducing factors, and is often hyper activated in many cancers including TNBCs. eIF4A is a potential therapeutic target in cancer, and inhibitors of eIF4A could be used in antiviral therapies and are being clinically tested for their anti-cancer potential. EIF4A2 regulates stem cell pluripotency during embryogenesis, and AUF1 regulates expression of multiple stem cell and metastasis-promoting transcription factors. CARP-1 KO cells also have diminished levels of genotoxic drug-induced EMT and metastasis-promoter Oct4. Further, RNA-seq analyses revealed significant down-regulation of epithelial mesenchymal transition, interferon α and γ response, and TNFα-NF-κB signaling pathways in all CARP-1 KO cells relative to their WT counterparts. These observations strongly suggest that CARP-1 promotes cancer cells growth, survival and EMT in part by regulating translation, expression, and signaling by EIF4A, AUF1, and NF-κB complexes. Homozygous deletion of CARP-1 is embryonic lethal at day 13.5 of embryogenesis, and CARP-1 -/- embryos fail to express EIF4A2 in dermal, mesenchymal and intercostal muscle cells. Although CARP-1 KO murine TNBC 4T1 and EO771 cells form tumors similar to their CARP-1 WT counterparts when subcutaneously xenografted in the female syngeneic mice, we interestingly noted reduced lung metastases in animals that had CARP-1 KO tumors when compared with animals with respective CARP-1 WT expressing tumors. Together with our findings that CARP-1 loss resulted in attenuation of EMT signaling, diminished EIF4A2 expression in mouse embryos as well as TNBC cells, our data underscore a novel pluripotency and tumor metastasis promoting function of CARP-1. Magesh Muthu, Sijana Dzinic, Hunter Dlugas, Jaganathan Venkatesh, Lisa A. Polin, Seongho Kim, Arun K. Rishi. CARP-1/CCAR1: A novel transducer of triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB469.