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.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

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.

Extracellular vesicle PD-L1 dynamics predict durable response to immune-checkpoint inhibitors and survival in patients with non-small cell lung cancer.

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.

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.

Targeting Wee1 kinase to suppress proliferation and survival of cisplatin-resistant head and neck squamous cell carcinoma.

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 IC50 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.

Translatome changes in acute myeloid leukemia cells post exposure to pegcrisantaspase and venetoclax.

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.

Phase I Clinical Trial of DNA Methyltransferase Inhibitor Decitabine and PARP Inhibitor Talazoparib Combination Therapy in Relapsed/Refractory Acute Myeloid Leukemia.

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.

DUOX2, a New Biomarker for Disseminated Gastric Cancer's Response to Low Dose Radiation in Mice.

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.

A novel XBP1 variant is highly enriched in cancer tissues and is specifically required for cancer cell survival.

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.

PP2A-activating Drugs Enhance FLT3 Inhibitor Efficacy through AKT Inhibition-Dependent GSK-3ß-Mediated c-Myc and Pim-1 Proteasomal Degradation.

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-3ß, and GSK-3ß inhibition blocked downregulation of both c-Myc and Pim-1 by PAD and FLT3 inhibitor cotreatment. GSK-3ß 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-3ß 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-3ß-mediated increased c-Myc and Pim-1 proteasomal degradation.

Antileukemic efficacy of a potent artemisinin combined with sorafenib and venetoclax.

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.

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.

SIRT3, a metabolic target linked to ataxia-telangiectasia mutated (ATM) gene deficiency in diffuse large B-cell lymphoma.

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.

Pharmacologic induction of innate immune signaling directly drives homologous recombination deficiency.

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.

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

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.

Pulsed focused ultrasound lowers interstitial fluid pressure and increases nanoparticle delivery and penetration in head and neck squamous cell carcinoma xenograft tumors.

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.

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.

Analysis of the Mechanisms of Action of Naphthoquinone-Based Anti-Acute Myeloid Leukemia Chemotherapeutics.

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.