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The ErbB/HER family of protein‑tyrosine kinases and PI3K represent crucial targets in the treatment of head and neck squamous cell carcinoma (HNSCC). A combination therapy of afatinib (ErbB inhibitor) and copanlisib (PI3K inhibitor), both Food and Drug Administration‑approved kinase inhibitors, can suppress the growth of human papillomavirus (HPV)‑positive HNSCC. The current study further evaluated the efficacy and clinical potential of this combination therapy for the treatment of HPV‑negative HNSCC in vitro and in vivo . Sulforhodamine B cell viability assay and Annexin V/propidium iodide staining demonstrated that this combination treatment markedly enhanced inhibition of cell viability and reduced cell survival when compared with treatment with either inhibitor alone in two HPV‑negative HNSCC cell lines. Notably, this combination also led to significant inhibition of xenograft tumor growth in mice, without any apparent effects on body weight. Western blot analysis found that copanlisib alone effectively blocked PI3K/Akt signaling but caused upregulation of HER2 and HER3 phosphorylation, as reported in other types of cancer. However, the combination of copanlisib and afatinib completely blocked phosphorylation of the ErbB family (including HER3) and Akt, while also increasing apoptosis. In conclusion, these results suggested that co‑targeting the ErbB family kinases and PI3K using a combination treatment of afatinib and copanlisib may have clinical potential for patients with HPV‑negative HNSCC. more...

Abstract: Glutamine dependency has been shown to be a metabolic vulnerability in acute myeloid leukemia (AML). Prior studies using several in vivo AML models showed that depletion of plasma glutamine, induced by long-acting crisantaspase (pegcrisantaspase [PegC]) was synergistic with the B-cell lymphoma-2 (BCL-2) inhibitor venetoclax (Ven), resulting in significantly reduced leukemia burden and enhanced survival. Here, we report a phase 1 study of the combination of Ven and PegC (VenPegC) for treating adult patients with relapsed or refractory AML, including patients who had previously received Ven. The primary end points were the incidence of regimen-limiting toxicities (RLTs) and the maximum tolerated dose (MTD). Twenty-five patients received at least 1 PegC dose with Ven, and 18 efficacy-evaluable patients completed at least 1 VenPegC cycle; 12 (67%) had previously received Ven. Hyperbilirubinemia was the RLT and occurred in 60% of patients treated with VenPegC; 20% had grade ≥3 bilirubin elevations. MTD was determined to be Ven 400 mg daily with biweekly PegC 750 IU/m2. The most common treatment-related adverse events of any grade in 25 patients who received VenPegC included antithrombin III decrease (52%), elevated transaminases (36%-48%), fatigue (28%), and hypofibrinogenemia (24%). No thromboembolic or hemorrhagic adverse events or clinical pancreatitis were observed. The overall complete remission rate in efficacy-evaluable patients was 33%. Response correlated with alterations in proteins involved in messenger RNA translation. In patients with RUNX1 mutations, the composite complete remission rate was 100%. This study was registered at www.ClinicalTrials.gov as #NCT04666649., (© 2025 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.) more...

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics., Methods: Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model., Results: Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes., Conclusions: Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways., (© 2024. The Author(s).) more...

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia -derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR- Cas9 -mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC 50 ) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic anti-proliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML., Competing Interests: AE has received research grants from Jazz Pharmaceuticals, NewLink Genetics, Amgen, and Servier Pharmaceuticals. AE is a global oncology advisory board member for Amgen and has served as an advisory board member for Genentech and Servier. AE and RL are Co-Founders and Scientific Advisors for KinaRx, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hameed, Bollino, Shetty, Carter-Cooper, Lapidus and Emadi.) more...

Background: Immune-checkpoint inhibitors (ICIs) are an effective therapeutic strategy, improving the survival of patients with lung cancer compared with conventional treatments. However, novel predictive biomarkers are needed to stratify which patients derive clinical benefit because the currently used and highly heterogenic histological PD-L1 has shown low accuracy. Liquid biopsy is the analysis of biomarkers in body fluids and represents a minimally invasive tool that can be used to monitor tumor evolution and treatment effects, potentially reducing biases associated with tumor heterogeneity associated with tissue biopsies. In this context, cytokines, such as transforming growth factor-β (TGF-β), can be found free in circulation in the blood and packaged into extracellular vesicles (EVs), which have a specific delivery tropism and can affect in tumor/immune system interaction. TGF-β is an immunosuppressive cytokine that plays a crucial role in tumor immune escape, treatment resistance, and metastasis. Thus, we aimed to evaluate the predictive value of circulating and EV TGF-β in patients with non-small-cell lung cancer receiving ICIs., Methods: Plasma samples were collected in 33 patients with advanced non-small-cell lung cancer before and during treatment with ICIs. EV were isolated from plasma by serial ultracentrifugation methods and circulating and EV TGF-β expression levels were evaluated by enzyme-linked immunosorbent assay., Results: Baseline high expression of TGF-β in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-β levels and tissue PD-L1 as a predictive biomarker., Conclusion: If validated, EV TGF-β could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-β blockade., Plain Language Summary: Treatment with immune-checkpoint inhibitors (ICIs) has improved the survival of some patients with lung cancer. However, the majority of patients do not benefit from this treatment, making it essential to develop more reliable biomarkers to identify patients most likely to benefit. In this pilot study, the expression of transforming growth factor-β (TGF-β) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-β before treatment were able to determine which patients would benefit from treatment with ICIs and have a longer survival with higher accuracy than circulating TGF-β and tissue PD-L1, which is the currently used biomarker in clinical practice., (© 2022 American Cancer Society.) more...

The impact of asparaginases on plasma asparagine and glutamine is well established. However, the effect of asparaginases, particularly those derived from Erwinia chrysanthemi (also called crisantaspase), on circulating levels of other amino acids is unknown. We examined comprehensive plasma amino acid panel measurements in healthy immunodeficient/immunocompetent mice as well as in preclinical mouse models of acute myeloid leukemia (AML) and pancreatic ductal adenocarcinoma (PDAC) using long-acting crisantaspase, and in an AML clinical study (NCT02283190) using short-acting crisantaspase. In addition to the expected decrease of plasma glutamine and asparagine, we observed a significant increase in plasma serine and glycine post-crisantaspase. In PDAC tumors, crisantaspase treatment significantly increased expression of serine biosynthesis enzymes. We then systematically reviewed clinical studies using asparaginase products to determine the extent of plasma amino acid reporting and found that only plasma levels of glutamine/glutamate and asparagine/aspartate were reported, without measuring other amino acid changes post-asparaginase. To the best of our knowledge, we are the first to report comprehensive plasma amino acid changes in mice and humans treated with asparaginase. As dysregulated serine metabolism has been implicated in tumor development, our findings offer insights into how leukemia/cancer cells may potentially overcome glutamine/asparagine restriction, which can be used to design future synergistic therapeutic approaches., Competing Interests: AE has received research grants from Jazz Pharmaceuticals, Amgen, NewLinks, and Servier. AE is a global oncology advisory board member for Amgen and has served as an advisory board member for Genentech, Servier, Kite Pharma, and Secura Bio. AE and RL are Co-Founders and Scientific Advisors for KinaRx, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bollino, Claiborne, Hameed, Ma, Tighe, Carter-Cooper, Lapidus, Strovel and Emadi.) more...

Objectives: To identify the most effective PI3K and EGFR inhibitors in HPV-positive head and neck squamous cell carcinoma (HNSCC) and investigate the efficacy of a combination of an ErbB family kinase inhibitor and a PI3K inhibitor to inhibit cell proliferation of HPV-positive HNSCC., Materials and Method: HPV-positive HNSCC cell lines were treated with the FDA approved ErbB kinase inhibitor, Afatinib or FDA-approved PI3K inhibitor, Copanlisib, alone or in combination, and phosphorylation and total protein levels of cells were assessed by Western blot analysis.Cell proliferation and apoptosis were examined by MTS assay, flow cytometry, and Western blots, respectively., Results: Copanlisib more effectively inhibited cell proliferation in comparison to other PI3K inhibitors tested. HPV-positive HNSCC cells differentially responded to cisplatin, Afatinib, or Copanlisib. The combination of Afatinib and Copanlisib more effectively suppressed cell proliferation and induced apoptosis compared to either treatment alone. Mechanistically, the combination of Afatinib and Copanlisib completely blocked phosphorylation of EGFR, HER2, HER3, and Akt as well as significantly decreased the HPV E7 expression compared to either treatment alone., Conclusion: Afatinib and Copanlisib more effectively suppress cell proliferation and survival of HPV-positive HNSCC in comparison to either treatment alone., (Copyright © 2022 Elsevier Ltd. All rights reserved.) more...

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies. more...

Background: Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs., Methods: Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively., Results: As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival., Conclusion: These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs., (© 2022. The Author(s).) more...

The clinical outcomes of patients with acute myeloid leukemia (AML) treated with available therapy remain unsatisfactory. We recently reported that the BCL-2 inhibitor venetoclax synergizes with pegcrisantaspase (Ven-PegC) and exhibits remarkable in vivo efficacy in a preclinical model of AML with complex karyotype. The Ven-PegC combination blocks synthesis of proteins in AML cells by inhibiting cap-dependent translation of mRNA. To further explore the impact of Ven-PegC on protein translation, we used polysome profiling and high-throughput RNA sequencing to characterize Ven-PegC-dependent changes to the translatome. Here we report that the translation of five mRNAs, including two microRNAs, one rRNA, and two mitochondrial genes, was altered after exposure to all three treatments (Ven, PegC, and Ven-PegC). We focused our translatome validation studies on six additional genes related to translational efficiency that were modified by Ven-PegC. Notably, Ven-PegC treatment increased the RNA translation and protein levels of Tribbles homologue 3 (TRIB3), eukaryotic translation initiation factor 3 subunit C (eIF3C), doublesex and mab-3-related transcription factor 1 (DMRT1), and salt-inducible kinase 1 (SIK1). We validated the observed changes in gene/protein expression in vitro and confirmed our cell line-based studies in the bone marrow of an AML patient-derived xenograft model after Ven-PegC treatment. These results support examining alterations in the translatome post chemotherapy to offer insight into the drug's mechanism of action and to inform future therapeutic decisions., Competing Interests: Conflict of interest disclosure AE has received research grants from Jazz Pharmaceuticals and NewLink Genetics. AE is a global oncology advisory board member for Amgen and has served as an advisory board member for Genentech, Servier, KiTE, and Secura Bio. AE and RGL are co-founders and scientific advisors for KinaRx, Inc. All other authors declare that they have no relevant competing interests., (Copyright © 2022 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.) more...

Purpose: Patients with acute myeloid leukemia (AML) unfit for, or resistant to, intensive chemotherapy are often treated with DNA methyltransferase inhibitors (DNMTi). Novel combinations may increase efficacy. In addition to demethylating CpG island gene promoter regions, DNMTis enhance PARP1 recruitment and tight binding to chromatin, preventing PARP-mediated DNA repair, downregulating homologous recombination (HR) DNA repair, and sensitizing cells to PARP inhibitor (PARPi). We previously demonstrated DNMTi and PARPi combination efficacy in AML in vitro and in vivo. Here, we report a phase I clinical trial combining the DNMTi decitabine and the PARPi talazoparib in relapsed/refractory AML., Patients and Methods: Decitabine and talazoparib doses were escalated using a 3 + 3 design. Pharmacodynamic studies were performed on cycle 1 days 1 (pretreatment), 5 and 8 blood blasts., Results: Doses were escalated in seven cohorts [25 patients, including 22 previously treated with DNMTi(s)] to a recommended phase II dose combination of decitabine 20 mg/m2 intravenously daily for 5 or 10 days and talazoparib 1 mg orally daily for 28 days, in 28-day cycles. Grade 3-5 events included fever in 19 patients and lung infections in 15, attributed to AML. Responses included complete remission with incomplete count recovery in two patients (8%) and hematologic improvement in three. Pharmacodynamic studies showed the expected DNA demethylation, increased PARP trapping in chromatin, increased γH2AX foci, and decreased HR activity in responders. γH2AX foci increased significantly with increasing talazoparib doses combined with 20 mg/m2 decitabine., Conclusions: Decitabine/talazoparib combination was well tolerated. Expected pharmacodynamic effects occurred, especially in responders., (©2022 American Association for Cancer Research.) more...

Purpose: We investigated the role of Wee1 kinase in cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines and determined the efficacy of either Wee1 inhibitor, AZD1775 alone, or in combination with cisplatin, on cisplatin-resistant HNSCC inhibition., Methods: Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell viability and apoptosis were examined by MTS assay and flow cytometry, respectively., Results: Wee1 kinase protein expression levels in five cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin-sensitive partners. Importantly, Wee1 knockdown inhibited cell proliferation and re-sensitized cells to cisplatin treatment. Interestingly, previous studies have also shown that Wee1 inhibitor AZD1775 synergizes with cisplatin to suppress cell proliferation of cisplatin-sensitive HNSCC. We found that AZD1775 inhibited both cisplatin-sensitive and resistant HNSCC with similar IC 50 values, which suggested that AZD1775 could overcome cisplatin resistance in cisplatin-resistant HNSCC. Mechanistically, AZD1775 and cisplatin cooperatively induced DNA damage and apoptosis., Conclusion: Wee1 inhibitor, AZD1775, and cisplatin coordinately suppressed proliferation and survival of HNSCC., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.) more...

Treatment options are rather limited for gastrointestinal cancer patients whose disease has disseminated into the intra-abdominal cavity. Here, we designed pre-clinical studies to evaluate the potential application of chemopotentiation by Low Dose Fractionated Radiation Therapy (LDFRT) for disseminated gastric cancer and evaluate the role of a likely biomarker, Dual Oxidase 2 (DUOX2). Nude mice were injected orthotopically with human gastric cancer cells expressing endogenous or reduced levels of DUOX2 and randomly assigned to four treatment groups: 1; vehicle alone, 2; modified regimen of docetaxel, cisplatin and 5'-fluorouracil (mDCF) for three consecutive days, 3; Low Dose- Whole Abdomen Radiation Therapy (LD-WART) (5 fractions of 0.15 Gy in three days), 4; mDCF and LD-WART. The combined regimen increased the odds of preventing cancer dissemination (mDCF + LD-WART OR = 4.16; 80% CI = 1.0, 17.29) in the DUOX2 positive tumors, while tumors expressing lower DUOX2 levels were more responsive to mDCF alone with no added benefit from LD-WART. The molecular mechanisms underlying DUOX2 effects in response to the combined regimen include NF-κB upregulation. These data are particularly important since our study indicates that about 33% of human stomach adenocarcinoma do not express DUOX2. DUOX2 thus seems a likely biomarker for potential clinical application of chemopotentiation by LD-WART. more...

XBP1 is a basic leucine zipper (bZIP) transcription factor and a key mediator of the endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR). XBP1-mediated transcription facilitates cell adaptation to ER stress and also promotes tumor progression, while suppressing anti-tumor immunity. Here we report a novel XBP1 variant, namely XBP1 variant 1 (XBP1v1, Xv1 for short), that is specifically required for survival of cancer cells. Xv1 contains a cryptic first exon that is conserved only in humans and great apes. Comparing to XBP1, Xv1 encodes a protein with a different N-terminal sequence containing 25 amino acids. Analysis of RNAseq database reveals that Xv1 is broadly expressed across cancer types but almost none in normal tissues. Elevated Xv1 expression is associated with poor survival of patients with several types of cancer. Knockdown of Xv1 induces death of multiple cancer cell lines but has little effect on non-cancerous cells in vitro. Moreover, knockdown of Xv1 also inhibits growth of a xenograft breast tumor in mice. Together, our results indicate that Xv1 is essential for survival of cancer cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.) more...

Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML. more...

Fms -like tyrosine-like kinase 3 internal tandem duplication (FLT3-ITD) is present in acute myeloid leukemia (AML) in 30% of patients and is associated with short disease-free survival. FLT3 inhibitor efficacy is limited and transient but may be enhanced by multitargeting of FLT3-ITD signaling pathways. FLT3-ITD drives both STAT5-dependent transcription of oncogenic Pim-1 kinase and inactivation of the tumor-suppressor protein phosphatase 2A (PP2A), and FLT3-ITD, Pim-1, and PP2A all regulate the c-Myc oncogene. We studied mechanisms of action of cotreatment of FLT3-ITD-expressing cells with FLT3 inhibitors and PP2A-activating drugs (PADs), which are in development. PADs, including FTY720 and DT-061, enhanced FLT3 inhibitor growth suppression and apoptosis induction in FLT3-ITD-expressing cell lines and primary AML cells in vitro and MV4-11 growth suppression in vivo PAD and FLT3 inhibitor cotreatment independently downregulated c-Myc and Pim-1 protein through enhanced proteasomal degradation. c-Myc and Pim-1 downregulation was preceded by AKT inactivation, did not occur in cells expressing myristoylated (constitutively active) AKT1, and could be induced by AKT inhibition. AKT inactivation resulted in activation of GSK-3β, and GSK-3β inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3β activation increased c-Myc proteasomal degradation through c-Myc phosphorylation on T58; infection with c-Myc with T58A substitution, preventing phosphorylation, blocked downregulation of c-Myc by PAD and FLT3 inhibitor cotreatment. GSK-3β also phosphorylated Pim-1L/Pim-1S on S95/S4. Thus, PADs enhance efficacy of FLT3 inhibitors in FLT3-ITD-expressing cells through a novel mechanism involving AKT inhibition-dependent GSK-3β-mediated increased c-Myc and Pim-1 proteasomal degradation., (©2021 American Association for Cancer Research.) more...

Artemisinins are active against human leukemia cell lines and have low clinical toxicity in worldwide use as antimalarials. Because multiagent combination regimens are necessary to cure fully evolved leukemias, we sought to leverage our previous finding that artemisinin analogs synergize with kinase inhibitors, including sorafenib (SOR), by identifying additional synergistic antileukemic drugs with low toxicity. Screening of a targeted antineoplastic drug library revealed that B-cell lymphoma 2 (BCL2) inhibitors synergize with artemisinins, and validation assays confirmed that the selective BCL2 inhibitor, venetoclax (VEN), synergized with artemisinin analogs to inhibit growth and induce apoptotic cell death of multiple acute leukemia cell lines in vitro. An oral 3-drug "SAV" regimen (SOR plus the potent artemisinin-derived trioxane diphenylphosphate 838 dimeric analog [ART838] plus VEN) killed leukemia cell lines and primary cells in vitro. Leukemia cells cultured in ART838 had decreased induced myeloid leukemia cell differentiation protein (MCL1) levels and increased levels of DNA damage-inducible transcript 3 (DDIT3; GADD153) messenger RNA and its encoded CCATT/enhancer-binding protein homologous protein (CHOP), a key component of the integrated stress response. Thus, synergy of the SAV combination may involve combined targeting of MCL1 and BCL2 via discrete, tolerable mechanisms, and cellular levels of MCL1 and DDIT3/CHOP may serve as biomarkers for action of artemisinins and SAV. Finally, SAV treatment was tolerable and resulted in deep responses with extended survival in 2 acute myeloid leukemia (AML) cell line xenograft models, both harboring a mixed lineage leukemia gene rearrangement and an FMS-like receptor tyrosine kinase-3 internal tandem duplication, and inhibited growth in 2 AML primagraft models., (© 2021 by The American Society of Hematology.) more...

Inactivation of Ataxia-telangiectasia mutated (ATM) gene results in an increased risk to develop cancer. We show that ATM deficiency in diffuse large B-cell lymphoma (DLBCL) significantly induce mitochondrial deacetylase sirtuin-3 (SIRT3) activity, disrupted mitochondrial structure, decreased mitochondrial respiration, and compromised TCA flux compared with DLBCL cells expressing wild type (WT)-ATM. This corresponded to enrichment of glutamate receptor and glutamine pathways in ATM deficient background compared to WT-ATM DLBCL cells. ATM -/- DLBCL cells have decreased apoptosis in contrast to radiosensitive non-cancerous A-T cells. In vivo studies using gain and loss of SIRT3 expression showed that SIRT3 promotes growth of ATM CRISPR knockout DLBCL xenografts compared to wild-type ATM control xenografts. Importantly, screening of DLBCL patient samples identified SIRT3 as a putative therapeutic target, and validated an inverse relationship between ATM and SIRT3 expression. Our data predicts SIRT3 as an important therapeutic target for DLBCL patients with ATM null phenotype. more...

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation., (© 2020 Wiley Periodicals LLC.) more...

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial., Competing Interests: Competing interest statement: F.V.R. and S.B.B. are co-inventors on US Provisional Patent Application Number 61/929,680 for the concept of the combinatorial therapy. more...

Nanocarriers offer a promising approach to significantly improve therapeutic delivery to solid tumors as well as limit the side effects associated with anti-cancer agents. However, their relatively large size can negatively affect their ability to efficiently penetrate into more interior tumor regions, ultimately reducing therapeutic efficacy. Poor penetration of large agents such as nanocarriers is attributed to factors in the tumor microenvironment such as elevated interstitial fluid pressure (IFP) and fibrillar collagen in the extracellular matrix. Our previous studies reported that pretreatment of solid tumor xenografts with nondestructive pulsed focused ultrasound (pFUS) can improve the delivery and subsequent therapy of a variety of therapeutic formulations in different tumor models, where the results were associated with expanded extracellular spaces (ECS), an increase in hydraulic conductivity, and decrease in tissue stiffness. Here, we demonstrate the inverse relationship between IFP and the penetration of systemically administered nanoparticle (NP) probes, where IFP increased from the tumor periphery to their center. Furthermore, we show that pretreatment with pFUS can safely reduce IFP and improve NP delivery; especially into the center of the tumors. These results coincide with effects generated in the fibrillar collagen network microstructure in the ECS as determined by quantitative polarized light microscopy. Whole tumor and histomorphometric analysis, however, did not show significant differences in collagen area fraction or collagen feature solidity, as well as tumor cross-sectional area and aspect ratio, as a result of the treatments. We present a biophysical model connecting the experimental results, where pFUS-mediated cytoarchitectural changes are associated with improved redistribution of the interstitial fluid and lower IFP. The resulting improvement in NP delivery supports our previous therapeutic studies and may have implications for clinical applications to improve therapeutic outcomes in cancer therapy. more...

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKβ in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKβ/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKβ, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKβ inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKβ in control of migration, invasion, and metastasis, and implicated that targeting IKKβ may be a potential therapy for cisplatin-resistant metastatic HNSCC. more...

Rho-associated protein kinases (ROCKs) are ubiquitously expressed in most adult tissues, and are involved in modulating the cytoskeleton, protein synthesis and degradation pathways, synaptic function, and autophagy to list a few. A few ROCK inhibitors, such as fasudil and netarsudil, are approved for clinical use. Here we present a new ROCK inhibitor, boronic acid containing HSD1590, which is more potent than netarsudil at binding to or inhibiting ROCK enzymatic activities. This compound exhibits single digit nanomolar binding to ROCK (Kds < 2 nM) and subnanomolar enzymatic inhibition profile (ROCK2 IC50 is 0.5 nM for HSD1590. Netarsudil, an FDA-approved drug, inhibited ROCK2 with IC50 = 11 nM under similar conditions). Whereas netarsudil was cytotoxic to breast cancer cell line, MDA-MB-231 (greater than 80% growth inhibition at concentrations greater than 5 μM), HSD1590 displayed low cytotoxicity to MDA-MB-231. Interestingly, at 1 μM HSD1590 inhibited the migration of MDA-MB-231 whereas netarsudil did not., (Copyright © 2019. Published by Elsevier Masson SAS.) more...

Acute myeloid leukemia (AML) is a neoplastic disorder resulting from clonal proliferation of poorly differentiated immature myeloid cells. Distinct genetic and epigenetic aberrations are key features of AML that account for its variable response to standard therapy. Irrespective of their oncogenic mutations, AML cells produce elevated levels of reactive oxygen species (ROS). They also alter expression and activity of antioxidant enzymes to promote cell proliferation and survival. Subsequently, selective targeting of redox homeostasis in a molecularly heterogeneous disease, such as AML, has been an appealing approach in the development of novel anti-leukemic chemotherapeutics. Naphthoquinones are able to undergo redox cycling and generate ROS in cancer cells, which have made them excellent candidates for testing against AML cells. In addition to inducing oxidative imbalance in AML cells, depending on their structure, naphthoquinones negatively affect other cellular apparatus causing neoplastic cell death. Here we provide an overview of the anti-AML activities of naphthoquinone derivatives, as well as analysis of their mechanism of action, including induction of reduction-oxidation imbalance, alteration in mitochondrial transmembrane potential, Bcl-2 modulation, initiation of DNA damage, and modulation of MAPK and STAT3 activity, alterations in the unfolded protein response and translocation of FOX-related transcription factors to the nucleus. more...

In the treatment of acute myeloid leukemia (AML), the "7 + 3"-based strategy, combining cytarabine 100-200 mg/m 2 for 7 days with an anthracycline for 3 days, remains the standard of care for younger and medically fit patients. Daunorubicin (DNR) and idarubicin (IDA) are the two anthracyclines most commonly used. DNR and IDA are used interchangeably with different conversion factors, as there is no high-level evidence on the equipotency of these two agents for AML treatment. To determine the equipotent doses of DNR and IDA, we first systematically reviewed studies directly comparing the clinical outcomes of AML induction therapy utilizing DNR and IDA. We found 15 articles that met our inclusion criteria and compared time-to-event survival end points as well as complete remission rates post-induction. The DNR:IDA equipotency ratio was estimated at 5.90 with 95% confidence interval (CI) 1.7-20.7. To validate the estimate from our meta-analysis biologically, we conducted in vitro tests comparing anti-AML activity of DNR and IDA against six AML cell lines and two primary AML cells from patients with different cytogenetic and molecular characteristics. Based on these in vitro data, the equipotency dose ratio between DNR and IDA was 4.06 with 95% CI 3.64-4.49. Combining the estimates from the meta-analysis and the in vitro data using inverse-variance weighting, the current best estimate of the DNR:IDA equipotent ratio is 4.1 with 95% CI 3.9-4.3. This estimate, however, is largely driven by the in vitro chemo-sensitivity data. Given clinical studies demonstrating the safety of IDA at higher doses, our work implies that dose intensification of IDA could be investigated in future clinical trials in AML. more...

Background: We investigated the role of the ETS-1 transcription factor in Head and Neck Squamous Cell Carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines., Methods: We examined its molecular link with SRC and MEK/ERK pathways and determined the efficacy of either MEK/ERK inhibitor PD0325901 or SRC inhibitor Dasatinib on cisplatin-resistant HNSCC inhibition., Results: We found that ETS-1 protein expression levels in a majority of cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin sensitive partners. High ETS-1 expression was also found in patient-derived, cisplatin-resistant HNSCC cells. While ETS-1 knockdown inhibited cell proliferation, migration, and invasion, it could still re-sensitize cells to cisplatin treatment. Interestingly, previous studies have shown that MER/ERK pathways could regulate ETS-1 through its phosphorylation at threonine 38 (T38). Although almost all cisplatin-resistant HNSCC cells we tested showed higher ETS-1 phosphorylation levels at T38, we found that inhibition of MEK/ERK pathways with the MEK inhibitor PD0325901 did not block this phosphorylation. In addition, treatment of cisplatin-resistant HNSCC cells with the MEK inhibitor completely blocked ERK phosphorylation but did not re-sensitize cells to cisplatin treatment. Furthermore, we found that, consistent with ETS-1 increase, SRC phosphorylation dramatically increased in cisplatin-resistant HNSCC, and treatment of cells with the SRC inhibitor, Dasatinib, blocked SRC phosphorylation and decreased ETS-1 expression. Importantly, we showed that Dasatinib, as a single agent, significantly suppressed cell proliferation, migration, and invasion, in addition to survival., Conclusions: Our results demonstrate that the SRC/ETS-1 pathway plays a crucial role and could be a key therapeutic target in cisplatin-resistant HNSCC treatment. more...

Expression of the receptor tyrosine kinase HER3 is negatively correlated with survival in ovarian cancer, and HER3 overexpression is associated with cancer progression and therapeutic resistance. Thus, improvements in HER3-targeted therapy could lead to significant clinical impact for ovarian cancer patients. Previous work from our group established multivalency as a potential strategy to improve the therapeutic efficacy of HER3-targeted ligands, including affibodies. Others have established HER3 affibodies as viable and potentially superior alternatives to monoclonal antibodies for cancer therapy. Here, bivalent HER3 affibodies were engineered for optimized production, specificity, and function as evaluated in an ovarian cancer xenograft model. Enhanced inhibition of HER3-mediated signaling and increased HER3 downregulation associated with multivalency could be achieved with a simplified construct, potentially increasing translational potential. Additionally, functional effects of affibodies due to multivalency were found to be specific to HER3 targeting, suggesting a unique molecular mechanism. Further, HER3 affibodies demonstrated efficacy in ovarian cancer xenograft mouse models, both as single agents and in combination with carboplatin. Overall, these results reinforce the potential of HER3-targeted affibodies for cancer therapy and establish treatment of ovarian cancer as an application where multivalent HER3 ligands may be useful. Further, this work introduces the potential of HER3 affibodies to be utilized as part of clinically relevant combination therapies (e.g., with carboplatin). more...

Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC), an aggressive and highly metastatic disease. Activation of eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1/2) play a critical role in the development, progression and metastasis of TNBC. Herein, we undertook a comprehensive study to evaluate the activity of a first-in-class Mnk1/2 protein degraders, racemic VNLG-152R and its two enantiomers (VNLG-152E1 and VNLG-152E2) in in vitro and in vivo models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader, superior to its pure enantiomers. By targeting Mnk1/2 protein degradation (activity), VNLG-152R potently inhibited both Mnk-eIF4E and mTORC1 signaling pathways and strongly regulated downstream factors involved in cell cycle regulation, apoptosis, pro-inflammatory cytokines/chemokines secretion, epithelial-mesenchymal transition (EMT) and metastasis. Most importantly, orally bioavailable VNLG-152R exhibited remarkable antitumor (91 to 100% growth inhibition) and antimetastatic (~80% inhibition) activities against cell line and patient-derived TNBC xenograft models, with no apparent host toxicity. Collectively, these studies demonstrate that targeting Mnk-eIF4E/mTORC1 signaling with a potent Mnk1/2 degrader, VNLG-152R, is a novel therapeutic strategy that can be developed as monotherapy for the effective treatment of patients with primary/metastatic TNBC. more...

Background: Acute myeloid leukemia (AML) remains one of the most lethal, rarely cured cancers, despite decades of active development of AML therapeutics. Currently, the 5-year survival of AML patients is about 30% and for elderly patients, the rate drops to <10%. About 30% of AML patients harbor an activating mutation in the tyrosine kinase domain (TKD) of Fms-Like Tyrosine kinase 3 (FLT3) or a FLT3 internal tandem duplication (FLT3-ITD). Inhibitors of FLT3, such as Rydapt that was recently approved by the FDA, have shown good initial response but patients often relapse due to secondary mutations in the FLT3 TKD, like D835Y and F691 L mutations., Methods: Alkynyl aminoisoquinoline and naphthyridine compounds were synthesized via Sonogashira coupling. The compounds were evaluated for their in vitro and in vivo effects on leukemia growth., Findings: The compounds inhibited FLT3 kinase activity at low nanomolar concentrations. The lead compound, HSN431, also inhibited Src kinase activity. The compounds potently inhibited the viability of MV4-11 and MOLM-14 AML cells with IC50 values <1 nM. Furthermore, the viability of drug-resistant AML cells harboring the D835Y and F691 L mutations were potently inhibited. In vivo efficacy studies in mice demonstrated that the compounds could drastically reduce AML proliferation in mice., Interpretation: Compounds that inhibit FLT3 and downstream targets like Src (for example HSN431) are good leads for development as anti-AML agents. FUND: Purdue University, Purdue Institute for Drug Discovery (PIDD), Purdue University Center for Cancer Research, Elks Foundation and NIH P30 CA023168., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.) more...

Background: Epidermal growth factor receptor (EGFR) plays an important role in head and neck squamous cell carcinoma (HNSCC) proliferation and therapy resistance, but the efficacy of targeting of EGFR for therapy has been limited. Here, we explore the molecular link between EGFR and inhibitor of κB kinase β/nuclear factor-κB (IKKβ/NF-κB) signalling pathways in the regulation of HNSCC EGFR inhibitor resistance., Methods: We performed in vitro experiments in eight human HNSCC cell lines and a patient-derived HNSCC cell line as well as in vivo xenografts in a human HNSCC cell line., Results: We found that treatment of all HNSCC cells with Gefitinib and Erlotinib, two Food Drug Administration-approved EGFR inhibitors, blocked the activity of Akt/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase, two crucial downstream effectors of EGFR, but up-regulated IKKβ/NF-κB signalling. In addition, induction of IKKβ/NF-κB by EGFR inhibitors required HER2 and HER3 expression. In keeping with these, IKKβ inhibitor CmpdA synergistically enhanced the efficacy of EGFR inhibitors to further inhibit in vitro HNSCC cell growth. Importantly, we demonstrated that the combination of Gefitinib with CmpdA inhibited xenograft tumour formation., Conclusion: Our data demonstrated that co-targeting EGFR and IKKβ with Gefitinib and IKKβ inhibitors could provide a potential novel therapy for head and neck squamous cell cancer. more...

Acute myeloid leukemia (AML) derives from the clonal expansion of immature myeloid cells in the bone marrow, and results in the disruption of normal hematopoiesis and subsequent bone marrow failure. The bone marrow microenvironment (BME) and its immune and other supporting cells are regarded to facilitate the survival, differentiation and proliferation of leukemia stem cells (LSCs), which enables AML cells to persist and expand despite treatment. Recent studies have identified epigenetic modifications among AML cells and BME constituents in AML, and have shown that epigenetic therapy can potentially reprogram these alterations. In this review, we summarize the interactions between the BME and LSCs, and discuss changes in how the BME and immune cells interact with AML cells. After describing the epigenetic modifications seen across chromatin, DNA, the BME, and the immune microenvironment, we explore how demethylating agents may reprogram these pathological interactions, and potentially re-sensitize AML cells to treatment., (© 2019 Elsevier Inc. All rights reserved.) more...

Human gamma delta T cells have extraordinary properties including the capacity for tumor cell killing. The major gamma delta T cell subset in human beings is designated Vγ9Vδ2 and is activated by intermediates of isoprenoid biosynthesis or aminobisphosphonate inhibitors of farnesyldiphosphate synthase. Activated cells are potent for killing a broad range of tumor cells and demonstrated the capacity for tumor reduction in murine xenotransplant tumor models. Translating these findings to the clinic produced promising initial results but greater potency is needed. Here, we review the literature on gamma delta T cells in cancer therapy with emphasis on the Vγ9Vδ2 T cell subset. Our goal was to examine obstacles preventing effective Vγ9Vδ2 T cell therapy and strategies for overcoming them. We focus on the potential for local activation of Vγ9Vδ2 T cells within the tumor environment to increase potency and achieve objective responses during cancer therapy. The gamma delta T cells and especially the Vγ9Vδ2 T cell subset, have the potential to overcome many problems in cancer therapy especially for tumors with no known treatment, lacking tumor-specific antigens for targeting by antibodies and CAR-T, or unresponsive to immune checkpoint inhibitors. Translation of amazing work from many laboratories studying gamma delta T cells is needed to fulfill the promise of effective and safe cancer immunotherapy. more...

Aim: Approximately 30% of acute myeloid leukemia (AML) patients carry FLT3 tyrosine kinase domain (TKD) mutations or internal tandem duplication (FLT3-ITD). Currently there is a paucity of compounds that are active against drug-resistant FLT3-ITD, which contains secondary mutations in the TKD, mainly at residues D835/F691., Results: HSD1169, a novel compound, is active against FLT3-ITD (D835 or F691). HSD1169 is also active against T-LAK cell-originated protein kinase (TOPK), a collaborating kinase that is highly expressed in AML cell lines. HSD1169 was active against MV4-11 and Molm-14 (FLT3-ITD cell lines) but not NOMO-1 or HL60 (FLT3-WT cell lines). HSD1169 was also active against sorafenib-resistant Molm13-res cell line (containing FLT3-ITD/D835Y)., Conclusion: HSD1169 or an analog could become a therapeutic agent for AML containing drug-resistant FLT3-ITD. more...

Depletion of glutamine (Gln) has emerged as a potential therapeutic approach in the treatment of acute myeloid leukemia (AML), as neoplastic cells require Gln for synthesis of cellular components essential for survival. Asparaginases deplete Gln, and asparaginase derived from Erwinia chrysanthemi (Erwinaze) appears to have the greatest glutaminase activity of the available asparaginases. In this Phase I study, we sought to determine the dose of Erwinaze that safely and effectively depletes plasma Gln levels to ≤ 120 μmol/L in patients with relapsed or refractory (R/R) AML. Five patients were enrolled before the study was halted due to issues with Erwinaze manufacturing supply. All patients received Erwinaze at a dose of 25,000 IU/m 2 intravenously three times weekly for 2 weeks. Median trough plasma Gln level at 48 h after initial Erwinaze administration was 27.6 μmol/L, and 80% (lower limit of 1-sided 95% CI 34%) of patients achieved at least one undetectable plasma Gln value (< 12.5 μmol/L), with the fold reduction (FR) in Gln level at 3 days, relative to baseline, being 0.16 (p < 0.001 for rejecting FR = 1). No dose-limiting toxicities were identified. Two patients responded, one achieved partial remission and one achieved hematologic improvement after six doses of Erwinaze monotherapy. These data suggest asparaginase-induced Gln depletion may have an important role in the management of patients with AML, and support more pharmacologic and clinical studies on the mechanistically designed asparaginase combinations in AML. more...

Purpose: fms -like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G 1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR ., (©2017 American Association for Cancer Research.) more...

The RUNX2 transcription factor promotes breast cancer growth and metastasis through interactions with a variety of cofactors that activate or repress target genes. Using a direct drug discovery approach we identified CADD522 as a small molecule that inhibits the DNA binding of the runt box domain protein, RUNX2. The current study defines the effect of CADD522 on breast cancer growth and metastasis, and addresses the mechanisms by which it exerts its anti-tumor activity. CADD522 treatment resulted in significant growth inhibition, clonogenic survival, tumorsphere formation, and invasion of breast cancer cells. CADD522 negatively regulated transcription of RUNX2 target genes such as matrix metalloproteinase-13, vascular endothelial growth factor and glucose transporter-1, but upregulated RUNX2 expression by increasing RUNX2 stability. CADD522 reduced RUNX2-mediated increases in glucose uptake and decreased the level of CBF-β and RUNX2 phosphorylation at the S451 residue. These results suggest several potential mechanisms by which CADD522 exerts an inhibitory function on RUNX2-DNA binding; interference with RUNX2 for the DNA binding pocket, inhibition of glucose uptake leading to cell cycle arrest, down-regulation of CBF-β, and reduction of S451-RUNX2 phosphorylation. The administration of CADD522 into MMTV-PyMT mice resulted in significant delay in tumor incidence and reduction in tumor burden. A significant decrease of tumor volume was also observed in a CADD522-treated human triple-negative breast cancer-patient derived xenograft model. CADD522 impaired the lung retention and outgrowth of breast cancer cells in vivo with no apparent toxicity to the mice. Therefore, by inhibiting RUNX2-DNA binding, CADD522 may represent a potential antitumor drug., Competing Interests: CONFLICTS OF INTEREST The authors disclose no potential conflicts of interest. more...

Aim: Mutated or overexpressed FLT3 drives about 30% of reported acute myeloid leukemia (AML). Currently, FLT3 inhibitors have shown durable clinical responses but a complete remission of AML with FLT3 inhibitors remains elusive due to mutation-driven resistance mechanisms. The development of FLT3 inhibitors that also target other downstream oncogenic kinases may combat the resistance mechanism., Results: 4-substituted aminoisoquinoline benzamides potently inhibit Src-family kinases and FLT3, including secondary mutations, such as FLT3D835. Modifications of aminoisoquinoline benzamide to aminoquinoline or aminoquinazoline abrogated FLT3 and Src-family kinase binding., Conclusion: The lead aminoisoquinolines potently inhibited FLT3-driven AML cell lines, MV4-11 and MOLM-14. These aminoisoquinoline benzamides represent new kinase scaffolds with high potential to be translated into anticancer agents. more...

Isocitrate dehydrogenase (IDH) mutations have been recognized in a few neoplasms including glioma, acute myeloid leukemia, chondrosarcoma, cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma. The direct methods to detect IDH mutations include DNA sequencing, immunohistochemistry (IHC), or by measuring its byproduct, 2-hydroxyglutarate (2-HG), in the blood or urine. Moreover, conventional magnetic resonance imaging can be modified to magnetic resonance spectroscopy (MRS) to measure 2-HG in tumor. By conducting a search in Medline/PubMed and ISI/Web of Science for the published articles in English related to the methods for detection of IDH mutations and its byproduct 2-HG, we compared different methodologies to detect these mutations and discuss advantages and limitations of each method. Studies in which a methodology of detection was compared with another modality were included. Multiple studies have shown that both DNA sequencing and IHC are reliable methods for detecting IDH mutations in glioma and other solid neoplasms. IHC appeared to be less costly, easier to perform, and may be slightly more accurate than DNA sequencing. 2-HG has also been measured in bone marrow aspirate, serum and urine of patients with mutant IDH acute myeloid leukemia, and correlated very well with sequencing and IHC. Lastly, in some glioma patients, MRS detected IDH mutations noninvasively and reliably with excellent correlations with other modalities such as IHC and sequencing. In conclusion, IHC, MRS, and 2-HG detection all are clinically useful and comparable with DNA sequencing in identifying IDH mutations in different neoplasms. 2-HG and MRS can be utilized for monitoring treatment response in a variety of neoplasms. more...

Acute myeloid leukemia (AML) is an aggressive malignancy with only a handful of therapeutic options. About 30% of AML patients harbor mutated FLT3 kinase, and thus, this cancer-driver has become a hotly pursued AML target. Herein we report a new class of FLT3 inhibitors, which potently inhibit the proliferation of acute myeloid leukemia (AML) cells at nanomolar concentrations. more...

Radiotherapy (RT) has long been known to be immunogenic. Mounting preclinical data demonstrate a synergistic anti-tumor effect when RT is used in combination with immune check point inhibitors (ICI). However, it is unclear how to best integrate RT with an ICI (i.e. dose fractionation, sequence, etc.). Here we explored the concept that RT delivered as an in situ tumor vaccine sequentially to separate tumors over time might stimulate more potent and rapid antitumor immune response than RT delivered to only one tumor. In essence, radiation to a second tumor could be likened to giving a vaccine "booster shot". Mice bearing pancreatic tumors in three different sites were injected with anti-PD-L1 antibody and exposed to three daily consecutive fractions of 4 Gy each at one or two sites with a one week interval. Our data indicate that delivering an RT to one tumor followed by an RT "booster shot" to a second tumor, compared to treating only one tumor with RT, significantly reduced tumor growth at a third non-irradiated site. This abscopal effect to the non-irradiated site was observed earlier (day 9) in mice that received RT to two tumors versusa single tumor (day 17). Decreased growth of the non-irradiated tumor correlated with a transient increase of the CD4/CD8 ratio in the tumor, increase myeloid-derived suppressor cells and tumor associated macrophages in the draining lymph nodes. These data warrant further exploration of sequentially treating multiple lesions with RT and ICI with the intent of generating a robust anti-tumor immune response. more...

The synthesis, characterization and antileukemic activity of rationally designed amino dimeric naphthoquinone (BiQ) possessing aziridine as alkylating moiety is described. Bis-aziridinyl BiQ decreased proliferation of acute myeloid leukemia (AML) cell lines and primary cells from patients, and exhibited potent (nanomolar) inhibition of colony formation and overall cell survival in AML cells. Effective production of reactive oxygen species (ROS) and double stranded DNA breaks (DSB) induced by bis-aziridinyl BiQ is reported. Bis-dimethylamine BiQ, as the isostere of bis-aziridinyl BiQ but without the alkylating moiety did not show as potent anti-AML activity. Systemic administration of bis-aziridinyl BiQ was well tolerated in NSG mice., (Copyright © 2016 Elsevier Ltd. All rights reserved.) more...

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer., Competing Interests: DISCLOSURES C.R., F.V.R. and S.B.B. share co-inventor ship on US Provisional Patent Application Number: 61/929,680 for the concept of the combinatorial therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.) more...

G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.) more...

NF-κB plays an important role in many types of cancer, including prostate cancer, but the role of the upstream kinase of NF-κB, IKKβ, in prostate cancer has neither been fully documented nor are there any effective IKKβ inhibitors used in clinical settings. Here, we have shown that IKKβ activity is mediated by multiple kinases including IKKα in human prostate cancer cell lines that express activated IKKβ. IHC analysis (IHC) of human prostate cancer tissue microarrays (TMA) demonstrates that phosphorylation of IKKα/β within its activation loop gradually increases in low to higher stage tumors as compared with normal tissue. The expression of cell proliferation and survival markers (Ki-67, Survivin) and epithelial-to-mesenchymal transition (EMT) markers (Slug, Snail), as well as cancer stem cell (CSC)-related transcription factors (Nanog, Sox2, Oct-4), also increase in parallel among the respective TMA samples analyzed. IKKβ, but not NF-κB, is found to regulate Nanog, which, in turn, modulates the levels of Oct4, Sox2, Snail, and Slug, indicating an essential role of IKKβ in regulating CSCs and EMT. The novel IKKβ inhibitor CmpdA inhibits constitutively activated IKKβ/NF-κB signaling, leading to induction of apoptosis and inhibition of proliferation, migration, and stemness in these cells. CmpdA also significantly inhibits tumor growth in xenografts without causing apparent in vivo toxicity. Furthermore, CmpdA and docetaxel act synergistically to inhibit proliferation of prostate cancer cells. These results indicate that IKKβ plays a pivotal role in prostate cancer, and targeting IKKβ, including in combination with docetaxel, may be a potentially useful strategy for treating advanced prostate cancer. Mol Cancer Ther; 15(7); 1504-14. ©2016 AACR., (©2016 American Association for Cancer Research.) more...

Described herein are our limited structure-activity relationship (SAR) studies on a 5:7-fused heterocycle (1), containing the 4,6,8-triaminoimidazo[4,5-e][1,3]diazepine ring system, whose synthesis and potent broad-spectrum anticancer activity we reported a few years ago. Our SAR efforts in this study are mainly focused on judicial attachment of substituents at N-1 and N(6)-positions of the heterocyclic ring. Our results suggest that there is some subtle correlation between the substituents attached at the N-1 position and those attached at the N(6)-position of the heterocycle. It is likely that there is a common hydrophobic binding pocket on the target protein that is occupied by the substituents attached at the N-1 and N(6)-positions of the heterocyclic ligand. This pocket appears to be large enough to hold either a C-18 alkyl chain of N(6) and no attachment at N-1, or a combined C-10 at N(6) and a CH2Ph at N-1. Any alkyl chain shorter or longer than C-10 at N(6) with a CH2Ph attached at N-1, would result in decrease of biological activity., (Copyright © 2016. Published by Elsevier Ltd.) more...

The overexpression or mutation of epidermal growth factor receptor (EGFR) has been associated with a number of cancers, including head and neck squamous cell carcinoma (HNSCC). Increasing evidence indicates that both the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of Rapamycin (mTOR) and the nuclear factor-kappa B (NF-κB) are constitutively active and contribute to aggressive HNSCC downstream of EGFR. However, whether these two oncogenic signaling pathways exhibit molecular and functional crosstalk in HNSCC is unclear. Our results now reveal that mTORC1, not mTORC2, contributes to NF-κB activation downstream of EGFR/PI3K/Akt signaling. Mechanistically, mTORC1 enhances the inhibitor of nuclear factor kappa-B kinase (IKK) activity to accelerate NF-κB signaling. Concomitantly, activated NF-κB/IKK up-regulates EGFR expression through positive feedback regulation. Blockage of NF-κB/IKK activity by the novel IKKβ specific inhibitor, CmpdA, leads to significant inhibition of cell proliferation and induction of apoptosis. CmpdA also sensitizes intrinsic cisplatin-resistant HNSCC cells to cisplatin treatment. Our findings reveal a new mechanism by which EGFR/PI3K/Akt/mTOR signaling promotes head and neck cancer progression and underscores the need for developing a therapeutic strategy for targeting IKK/NF-κB either as a single agent or in combination with cisplatin in head and neck cancer., Competing Interests: No potential conflicts of interest were disclosed. more...

Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients. All compounds possess one halogen and one hydroxyl group on the quinone cores. Dimeric, but not monomeric, naphthoquinones demonstrated significant anti-AML activity in the cell lines and primary cells from patients with favorable therapeutic index compared to normal hematopoietic cells. BiQ-1 effectively inhibited clonogenicity and induced apoptosis as measured by Western blotting and Annexin V staining and mitochondrial membrane depolarization by flow cytometry. BiQ-1 significantly enhances intracellular ROS levels in AML cells and upregulates expression of key anti-oxidant protein, Nrf2. Notably, systemic exposure to BiQ-1 was well tolerated in mice. In conclusion, we propose that BiQ-induced therapeutic augmentation of ROS in AML cells with dysregulation of antioxidants kill leukemic cells while normal cells remain relatively intact. Further studies are warranted to better understand this class of potential chemotherapeutics. more...