Long-acting Erwinia chrysanthemi, Pegcrisantaspase, induces alternate amino acid biosynthetic pathways in a preclinical model of pancreatic ductal adenocarcinoma.

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.

Dual targeting of glutamine and serine metabolism in acute myeloid leukemia.

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 (IC50) 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.

Baseline extracellular vesicle TGF-ß is a predictive biomarker for response to immune checkpoint inhibitors and survival in non-small cell lung cancer.

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-ß (TGF-ß), 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-ß 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-ß 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-ß expression levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: Baseline high expression of TGF-ß in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-ß levels and tissue PD-L1 as a predictive biomarker. CONCLUSION: If validated, EV TGF-ß could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-ß 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-ß (TGF-ß) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-ß 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-ß and tissue PD-L1, which is the currently used biomarker in clinical practice.

Erwinia asparaginase (crisantaspase) increases plasma levels of serine and glycine.

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.

Simultaneously targeting ErbB family kinases and PI3K in HPV-positive head and neck squamous cell carcinoma.

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.

Pre-Clinical Activity of Amino-Alcohol Dimeric Naphthoquinones as Potential Therapeutics for Acute Myeloid Leukemia.

BACKGROUND: The clinical outcomes of patients with Acute Myeloid Leukemia (AML) remain unsatisfactory. Therefore the development of more efficacious and better-tolerated therapy for AML is critical. We have previously reported anti-leukemic activity of synthetic halohydroxyl dimeric naphthoquinones (BiQ) and aziridinyl BiQ. OBJECTIVE: This study aimed to improve the potency and bioavailability of BiQ compounds and investigate antileukemic activity of the lead compound in vitro and a human AML xenograft mouse model. METHODS: We designed, synthesized, and performed structure-activity relationships of several rationally designed BiQ analogues with amino alcohol functional groups on the naphthoquinone core rings. The compounds were screened for anti-leukemic activity and the mechanism as well as in vivo tolerability and efficacy of our lead compound was investigated. RESULTS: We report that a dimeric naphthoquinone (designated BaltBiQ) demonstrated potent nanomolar anti-leukemic activity in AML cell lines. BaltBiQ treatment resulted in the generation of reactive oxygen species, induction of DNA damage, and inhibition of indoleamine dioxygenase 1. Although BaltBiQ was tolerated well in vivo, it did not significantly improve survival as a single agent, but in combination with the specific Bcl-2 inhibitor, Venetoclax, tumor growth was significantly inhibited compared to untreated mice. CONCLUSION: We synthesized a novel amino alcohol dimeric naphthoquinone, investigated its main mechanisms of action, reported its in vitro anti-AML cytotoxic activity, and showed its in vivo promising activity combined with a clinically available Bcl-2 inhibitor in a patient-derived xenograft model of AML.

Venetoclax and pegcrisantaspase for complex karyotype acute myeloid leukemia.

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.

Suppression of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma through IKKß inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß 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 IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß 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 IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.

Potently inhibiting cancer cell migration with novel 3H-pyrazolo[4,3-f]quinoline boronic acid ROCK inhibitors.

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.

Regulation of cisplatin-resistant head and neck squamous cell carcinoma by the SRC/ETS-1 signaling pathway.

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.

Equipotent doses of daunorubicin and idarubicin for AML: a meta-analysis of clinical trials versus in vitro estimation.

In the treatment of acute myeloid leukemia (AML), the "7 + 3"-based strategy, combining cytarabine 100-200 mg/m2 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.

Amino alkynylisoquinoline and alkynylnaphthyridine compounds potently inhibit acute myeloid leukemia proliferation in mice.

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.

Dual FLT3/TOPK inhibitor with activity against FLT3-ITD secondary mutations potently inhibits acute myeloid leukemia cell lines.

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.

Asparaginase Erwinia chrysanthemi effectively depletes plasma glutamine in adult patients with relapsed/refractory acute myeloid leukemia.

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/m2 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.

Identification of New FLT3 Inhibitors That Potently Inhibit AML Cell Lines via an Azo Click-It/Staple-It Approach.

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.

Aminoisoquinoline benzamides, FLT3 and Src-family kinase inhibitors, potently inhibit proliferation of acute myeloid leukemia cell lines.

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.

Synthesis, characterization and antineoplastic activity of bis-aziridinyl dimeric naphthoquinone - A novel class of compounds with potent activity against acute myeloid leukemia cells.

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.

Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities.

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.

Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2.

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.

Perturbation of cellular oxidative state induced by dichloroacetate and arsenic trioxide for treatment of acute myeloid leukemia.

The incidence of acute myeloid leukemia (AML) is rising and the outcome of current therapy, which has not changed significantly in the last 40 years, is suboptimal. Cellular oxidative state is a credible target to selectively eradicate AML cells, because it is a fundamental property of each cell that is sufficiently different between leukemic and normal cells, yet its aberrancy shared among different AML cells. To this end, we tested whether a short-time treatment of AML cells, including cells with FLT3-ITD mutation, with sub-lethal dose of dichloroacetate (DCA) (priming) followed by pharmacologic dose of arsenic trioxide (ATO) in presence of low-dose DCA could produce insurmountable level of oxidative damage that kill AML cells. Using cellular cytotoxicity, apoptotic and metabolic assays with both established AML cell lines and primary AML cells, we found that priming with DCA significantly potentiated the cytotoxicity of ATO in AML cells in a synergistic manner. The combination decreased the mitochondrial membrane potential as well as expression of Mcl-1 and GPx in primary AML cells more than either drug alone. One patient with AML whose disease was refractory to several lines of prior treatments was treated with this combination, and tolerated it well. These data suggest that targeting cellular redox balance in leukemia may provide a therapeutic option for AML patients with relapsed/refractory disease.