A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML.

Improvement in survival has been achieved for children and adolescents with AML but is largely attributed to enhanced supportive care as opposed to the development of better treatment regimens. High risk subtypes continue to have poor outcomes with event free survival rates <40% despite the use of high intensity chemotherapy in combination with hematopoietic stem cell transplant. Here we combine high-throughput screening, intracellular accumulation assays, and in vivo efficacy studies to identify therapeutic strategies for pediatric AML. We report therapeutics not currently used to treat AML, gemcitabine and cabazitaxel, have broad anti-leukemic activity across subtypes and are more effective relative to the AML standard of care, cytarabine, both in vitro and in vivo. JAK inhibitors are selective for acute megakaryoblastic leukemia and significantly prolong survival in multiple preclinical models. Our approach provides advances in the development of treatment strategies for pediatric AML.

Therapeutic Targeting of KDM1A/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response.

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 -1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 µmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902-16. ©2018 AACR.

Hypoxia-induced upregulation of BMX kinase mediates therapeutic resistance in acute myeloid leukemia.

Oncogenic addiction to the Fms-like tyrosine kinase 3 (FLT3) is a hallmark of acute myeloid leukemia (AML) that harbors the FLT3-internal tandem duplication (FLT3-ITD) mutation. While FLT3 inhibitors like sorafenib show initial therapeutic efficacy, resistance rapidly develops through mechanisms that are incompletely understood. Here, we used RNA-Seq-based analysis of patient leukemic cells and found that upregulation of the Tec family kinase BMX occurs during sorafenib resistance. This upregulation was recapitulated in an in vivo murine FLT3-ITD-positive (FLT3-ITD+) model of sorafenib resistance. Mechanistically, the antiangiogenic effects of sorafenib led to increased bone marrow hypoxia, which contributed to HIF-dependent BMX upregulation. In in vitro experiments, hypoxia-dependent BMX upregulation was observed in both AML and non-AML cell lines. Functional studies in human FLT3-ITD+ cell lines showed that BMX is part of a compensatory signaling mechanism that promotes AML cell survival during FLT3 inhibition. Taken together, our results demonstrate that hypoxia-dependent upregulation of BMX contributes to therapeutic resistance through a compensatory prosurvival signaling mechanism. These results also reveal the role of off-target drug effects on tumor microenvironment and development of acquired drug resistance. We propose that the bone marrow niche can be altered by anticancer therapeutics, resulting in drug resistance through cell-nonautonomous microenvironment-dependent effects.

OCTN1 Is a High-Affinity Carrier of Nucleoside Analogues.

Resistance to xenobiotic nucleosides used to treat acute myeloid leukemia (AML) and other cancers remains a major obstacle to clinical management. One process suggested to participate in resistance is reduced uptake into tumor cells via nucleoside transporters, although precise mechanisms are not understood. Through transcriptomic profiling, we determined that low expression of the ergothioneine transporter OCTN1 (SLC22A4; ETT) strongly predicts poor event-free survival and overall survival in multiple cohorts of AML patients receiving treatment with the cytidine nucleoside analogue cytarabine. Cell biological studies confirmed OCTN1-mediated transport of cytarabine and various structurally related cytidine analogues, such as 2'deoxycytidine and gemcitabine, occurs through a saturable process that is highly sensitive to inhibition by the classic nucleoside transporter inhibitors dipyridamole and nitrobenzylmercaptopurine ribonucleoside. Our findings have immediate clinical implications given the potential of the identified transport system to help refine strategies that could improve patient survival across multiple cancer types where nucleoside analogues are used in cancer treatment. Cancer Res; 77(8); 2102-11. ©2017 AACR.

Evaluation of artemisinins for the treatment of acute myeloid leukemia.

PURPOSE: Investigate antileukemic activity of artemisinins, artesunate (ART), and dihydroartemisinin (DHA), in combination with cytarabine, a key component of acute myeloid leukemia (AML) chemotherapy using in vitro and in vivo models. METHODS: Using ten human AML cell lines, we conducted a high-throughput screen to identify antimalarial agents with antileukemic activity. We evaluated effects of ART and DHA on cell viability, cytotoxicity, apoptosis, lysosomal integrity, and combination effects with cytarabine in cell lines and primary patient blasts. In vivo pharmacokinetic studies and efficacy of single-agent ART or combination with cytarabine were evaluated in three xenograft models. RESULTS: ART and DHA had the most potent activity in a panel of AML cell lines, with selectivity toward samples harboring MLL rearrangements and FLT3-ITD mutations. Combination of ART or DHA was synergistic with cytarabine. Single-dose ART (120 mg/kg) produced human equivalent exposures, but multiple dose daily administration required for in vivo efficacy was not tolerated. Combination treatment produced initial regression, but did not prolong survival in vivo. CONCLUSIONS: The pharmacology of artemisinins is problematic and should be considered in designing AML treatment strategies with currently available agents. Artemisinins with improved pharmacokinetic properties may offer therapeutic benefit in combination with conventional therapeutic strategies in AML.

Urinary angiostatin levels are elevated in patients with epithelial ovarian cancer.

OBJECTIVE: The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC. METHODS: VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. RESULTS: AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements. CONCLUSIONS: Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted.

Expression of Semaphorin 3F and Its Receptors in Epithelial Ovarian Cancer, Fallopian Tubes, and Secondary Müllerian Tissues.

While semaphorins and their receptors appear to play a role in tumor carcinogenesis, little is known about the role of semaphorin 3F (S3F) in epithelial ovarian cancer (EOC) development. Therefore, we sought to determine the clinical relationship between S3F and its receptors, neuropilin-2 (NP-2) and neuropilin-1 (NP-1) with EOC progression. We analyzed the immunohistological expression of S3F, NP-2, and NP-1 in clinical specimens of normal ovaries (N), benign cystadenomas (Cy), well-differentiated adenocarcinomas (WD), poorly-differentiated adenocarcinomas (PD), inclusion cysts (IC), paraovarian cysts (PC), and fallopian tubes (FT). Tissue sections were evaluated for staining intensity and percentage of immunoreactive epithelia. We found that expression of S3F and NP-2 decreased while NP-1 expression increased with EOC progression. Interestingly, we also found elevated expression of S3F, NP-2, and NP-1 in epithelia of ICs, PCs, and FT. Our findings indicate that loss or deregulation of semaphorin signaling may play an important role in EOC development.