Low Nanomolar Doses Research Articles

Clickable Shiga Toxin B Subunit for Drug Delivery in Cancer Therapy.

In recent years, receptor-mediated drug delivery has gained major attention in the treatment of cancer. The pathogen-derived Shiga Toxin B subunit (STxB) can be used as a carrier that detects the tumor-associated glycosphingolipid globotriaosylceramide (Gb3) receptors. While drug conjugation via lysine or cysteine offers random drug attachment to carriers, click chemistry has the potential to improve the engineering of delivery systems as the site specificity can eliminate interference with the active binding site of tumor ligands. We present the production of recombinant STxB in its wild-type (STxBwt) version or incorporating the noncanonical amino acid azido lysine (STxBAzK). The STxBwt and STxBAzK were manufactured using a growth-decoupled Escherichia coli (E. coli)-based expression strain and analyzed via flow cytometry for Gb3 receptor recognition and specificity on two human colorectal adenocarcinoma cell lines-HT-29 and LS-174-characterized by high and low Gb3 abundance, respectively. Furthermore, STxBAzK was clicked to the antineoplastic agent monomethyl auristatin E (MMAE) and evaluated in cell-killing assays for its ability to deliver the drug to Gb3-expressing tumor cells. The STxBAzK-MMAE conjugate induced uptake and release of the MMAE drug in Gb3-positive tumor cells, reaching 94% of HT-29 cell elimination at 72 h post-treatment and low nanomolar doses while sparing LS-174 cells. STxBAzK is therefore presented as a well-functioning drug carrier, with a possible application in cancer therapy. This research demonstrates the feasibility of lectin carriers used in delivering drugs to tumor cells, with prospects for improved cancer therapy in terms of straightforward drug attachment and effective cancer cell elimination.

Pan-HDAC inhibitors augment IL2-induced proliferation of NK cells via the JAK2-STAT5B signaling pathway

BackgroundNatural killer (NK) cells are a subtype of lymphocytes with the ability to quickly and efficiently identify and eliminate tumor cells. In the presence of IL2, NK cells can divide rapidly but in limited numbers. According to previous studies, in vivo treatment with histone deacetylase (HDAC) inhibitors did not impair NK-cell function. This study aimed to investigate the effect of HDAC inhibitors on NK-cell proliferation and the underlying regulatory mechanism. MethodsNK92 cells, primary NK (pNK) cells, and CD19-CAR-NK92 cells were treated with low concentrations of pan-HDACi Dacinostat (Dac) and Panobinostat (Pan) in the presence of IL2, and Cell Counting Kit-8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry assays were used to assess cell proliferation and apoptosis. The expression of granzyme B was detected by immunofluorescence, and the expression of CD107a and NKG2D was determined by flow cytometry. The downstream regulatory genes were identified by RNA-seq, and the “JAK-STAT signaling pathway”- and “Cell cycle signaling pathway”-related genes were detected by real-time quantitative polymerase chain reaction (RT–qPCR) and Western blot analysis. The JAK2V617F mouse model was constructed to simulate the upregulation of the JAK2 signaling pathway in vivo, and the NK proliferation was evaluated by flow cytometry. A tumor-bearing nude mouse model was constructed to determine the anti-tumor efficacy of NK92 cells following Dac treatment. ResultsIn the presence of IL2, the proliferation rate of NK92 cells, pNK cells, and CD19-CAR-NK92 cells treated with pan-HDACi Dac and Pan at low nanomolar doses was significantly increased, although cell function was unaffected. Low doses of Dac upregulated the JAK-STAT signaling pathway and enhance the cell cycle via that pathway. In addition, the in vivo experiment in nude mice showed that the capacity of Dac treated NK92 cells to eliminate tumor cells was unaffected. ConclusionLow nanomolar doses of Pan-HDACi enhanced IL2-induced NK cell proliferation without compromising the functioning of NK cells.

Combination of the Kdm-Family Inhibitor, Jib-04, Improves Response to Acute Myeloid Leukemia Cells to Venetoclax

Background: Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy with a 5-year survival rate of about 30% in adults with AML. While over the prior several decades, the standard of care treatment for AML changed very little, a number of novel targeted therapies have emerged in recent years. Promising results from clinical trials with the BCL2-specific inhibitor, Venetoclax, in combination with hypomethylating agents (HMA) or low-dose cytarabine for AML treatment have generated enthusiasm in the field. However, data from a phase II trial indicated a 30% failure rate after Venetoclax+HMA treatment of newly diagnosed patients, suggesting additional options are needed. Thus, we sought to explore alternative drug combinations that may enhance the efficacy of Venetoclax. JIB-04 is a potent small molecule inhibitor of multiple members of the histone lysine demethylase (KDM) 4, 5 and 6 families with KDM5A being the most sensitive. Expression levels of histone lysine-specific demethylases are higher in AML compared to normal bone marrow, suggesting they are potential targets for therapeutic intervention. Pharmacological inhibition or genetic knock down of KDM 4, 5 and 6 family members leads to reduced proliferation, cell cycle arrest, and increased apoptosis of AML cells of various subtypes. These preliminary findings compelled us to explore the anti-leukemic activity of JIB-04 alone and in combination with Venetoclax. Methods: We treated AML cell lines, patient AML blasts, and CD34+ cells from healthy donors with JIB-04, Venetoclax, or the combination for 72 hours in vitro. Apoptosis was quantified by AnnexinV/propidium iodide (PI) staining. Cell proliferation of AML cell lines treated with JIB-04 was evaluated using MTT assay and transcriptome profiling was done by RNA-Seq. NSG mice were injected with MOLM-13 cells and treated with JIB-04, Venetoclax or the combination for two weeks. For the knock-down (KD) of DDIT4 MOLM-13 cells were transduced with a shRNA and cells were treated with Venetoclax for 72h and analyzed for apoptosis. Results: Treatment of AML cell lines with low nanomolar doses (IC50 range from 8-60nM) of JIB-04 resulted in inhibition of proliferation and induction of apoptosis. In all AML cell lines tested, including AML cell lines with relative resistance to Venetoclax, the combination of JIB-04 with Venetoclax showed a strong synergistic effect. Moreover, patient AML blasts also showed a synergistic response to the drug combination compared to single-drug treatments (Figure 1a). In contrast, viability of normal CD34+ hematopoietic cells was not affected by JIB-04 treatment even up to a dose of 100nM that leads to apoptosis in AML cells, indicating a broad therapeutic window. The combination treatment of NSG mice injected with MOLM-13 cells resulted in reduced engraftment of huCD45+ cells compared to the single treatments in the blood, bone marrow and spleen. RNA sequencing of AML-OCI3 and MOLM-13 cells treated with JIB-04 revealed regulation of genes related to the mTOR pathway, stress response or amino acid metabolism, indicating possible mechanisms affecting proliferation and apoptosis. DDIT4 (REDD1), a regulator of the mTOR pathway, was downregulated after JIB-04 treatment in both cell lines. KD of DDIT4 in MOLM-13 cells resulted in increased sensitivity to Venetoclax (Figure 1b). Summary/Conclusion: While the epigenetic inhibitor JIB-04 alone shows modest anti-leukemic effect, this study highlights the potential therapeutic benefit of combining JIB-04 and Venetoclax in AML. The analysis of transcriptomic changes after JIB-04 treatment identified the mTOR pathway regulator DDIT4 as a likely mediator of the sensitization of AML cells to Venetoclax. High DDIT4 expression is associated with a worse outcome in AML making it an attractive therapeutic target for AML, especially in combination with Venetoclax. Our result would suggest this drug combination as a possible treatment option for AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Interactions between cannabinoid and opioid receptors in a mouse model of diabetic neuropathy.

Diabetic neuropathy, often associated with diabetes mellitus, is a painful condition with no known effective treatment except glycemic control. Studies with neuropathic pain models report alterations in cannabinoid and opioid receptor expression levels; receptors whose activation induces analgesia. We examined whether interactions between CB1R and opioid receptors could be targeted for the treatment of diabetic neuropathy. For this, we generated antibodies that selectively recognize native CB1R-MOR and CB1R-DOR heteromers using a subtractive immunization strategy. We assessed the levels of CB1R, MOR, DOR, and interacting complexes using a model of streptozotocin-induced diabetic neuropathy and detected increased levels of CB1R, MOR, DOR, and CB1R-MOR complexes compared with those in controls. An examination of G-protein signaling revealed that activity induced by the MOR, but not the DOR agonist, was potentiated by low nanomolar doses of CB1R ligands, including antagonists, suggesting an allosteric modulation of MOR signaling by CB1R ligands within CB1R-MOR complexes. Because the peptide endocannabinoid, hemopressin, caused a significant potentiation of MOR activity, we examined its effect on mechanical allodynia and found that it blocked allodynia in wild-type mice and mice with diabetic neuropathy lacking DOR (but have CB1R-MOR complexes). However, hemopressin does not alter the levels of CB1R-MOR complexes in diabetic mice lacking DOR but increases the levels of CB1R-DOR complexes in diabetic mice lacking MOR. Together, these results suggest the involvement of CB1R-MOR and CB1R-DOR complexes in diabetic neuropathy and that hemopressin could be developed as a potential therapeutic for the treatment of this painful condition.

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells.

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration-approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30-100 nm) ERBB2- EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs.

Truncated Actin-Targeting Macrolide Derivative Blocks Cancer Cell Motility and Invasion of Extracellular Matrix.

Cancer metastasis is a complex process involving highly motile tumor cells that breach tissue barriers, enter the bloodstream and lymphatic system, and disseminate throughout the body as circulating tumor cells. The primary cellular mechanism contributing to these critical events is the reorganization of the actin cytoskeleton. Mycalolide B (MycB) is an actin-targeting marine macrolide that can suppress proliferation, migration, and invasion of breast and ovarian cancer cells at low nanomolar doses. Through structure-activity relationship studies focused on the actin-binding tail region (C24-C35) of MycB, we identified a potent truncated derivative that inhibits polymerization of G-actin and severs F-actin by binding to actin's barbed end cleft. Biological analyses of this miniature MycB derivative demonstrate that it causes a rapid collapse of the actin cytoskeleton in ovarian cancer cells and impairs cancer cell motility and invasion of the extracellular matrix (ECM) by inhibiting invadopodia-mediated ECM degradation. These studies provide essential proof-of-principle for developing actin-targeting therapeutic agents to block cancer metastasis and establish a synthetically tractable barbed end-binding pharmacophore that can be further improved by adding targeting groups for precision drug design.

Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.

Dual Targeting of Mitochondrial Vulnerability Using Complex I Inhibitor Iacs-010759 with Bcl-2 Inhibitor Venetoclax and Azacitidine in Pre-Clinical Acute Myeloid Leukemia (AML) Models

Despite recent approval of hypomethylating agent/venetoclax (HMA+VEN) therapy for older patients (pt) with AML unfit for induction chemotherapy, their outcomes remain suboptimal. Such pts have a median overall survival of only 14 months and only approximately 35% of pts enjoying long-term survival (DiNardo, EHA 2020). Approximately 20-40% of newly diagnosed older pts with AML do not respond to the HMA+VEN regimens, with higher rates of refractory disease as well as early relapse in high-risk pts. Metabolic reprogramming and dependence on mitochondrial oxidative phosphorylation (OxPhos) is a core feature of AML leukemia stem cells (LSC). Recent reports have shown that upregulation of OxPhos confers intrinsic and acquired resistance to VEN in AML, multiple myeloma and lymphoid malignancies, which can be reversed by disrupting OxPhos; mutated TP53 has been shown to confer intrinsic resistance to venetoclax through increased OxPhos (Nechiporuk T, et al. Cancer Discovery 2019). We hypothesized that combined blockade of mitochondrial fitness by OxPhos inhibitors and of BCL-2 with VEN/azacitidine (AZA) will perform synergistically in pre-clinical AML models. To test this hypothesis, we utilized a novel complex I inhibitor IACS-010759 that effectively inhibits cell respiration and leukemia progression in the in vitro and in vivo AML pre-clinical models (Molina, Nat Med 2018). Priming of MOLM-13 cells with 20nM VEN for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while single agents reduced OCR by <15%. This translated into accelerated loss of the mitochondrial membrane potential measured by JC-1 flow cytometry, cleavage of caspase 3 and synergistic reduction (>70%) of viable cell numbers in several AML cell lines tested (OCI-AML2, MV-4-11, and MOLM-13) (Figure 1). By co-immunoprecipitation VEN disrupted interaction of BCL-2 with the mitochondrial protein VDAC, known to regulate ADP/ATP exchange during electron transport across mitochondria membrane; this resulted in dramatic reduction of the intracellular ATP and CTP levels measured by MS-based metabolomics. Further, VEN increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of VEN and IACS-010759, possibly because of RNA degradation. In primary AML samples (n=3) and AML PDX cells (n=4) cultured ex vivo, combined VEN and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. We next tested the efficacy of the "triple" combination of VEN, AZA and IACS-010759 in the in vivo AML PDX models. We injected AML PDX cells 3871344 (with no mutations identified by targeted sequencing) and 4404778 (harboring IDH1, NPM1, FLT3-ITD mutations) into NRG mice and upon engraftment, randomized mice into 4 groups to receive 2 cycles of treatment with 3 weeks interruption between cycles: vehicle, VEN (50mg/kg daily, 5 days on/2 days off, day 1-21) with AZA (2.5 mg/kg daily, day 1-7), IACS-010759 (1mg/kg ?daily, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. In the less aggressive model 3871344, all therapies reduced circulating leukemia burden, and the triple treatment achieved best efficacy, with average circulating tumor burden at 10 weeks after cycle 2 of 90.8%, 38.7%, 68.8% and 7.4% in vehicle, VEN+AZA, IACS-010759, and triple-therapy cohorts, respectively. In the aggressive model 4404778, the treatments were less effective, but the combination offered highest activity, with average circulating tumor burden of 71.0%, 52.3%, 88.7% and 39.9% in vehicle, IACS-010759, VEN+AZA and triple-therapy cohorts, respectively (Figure 1). Analysis of survival and additional PDX models are ongoing and will be reported. In summary, our study demonstrates that BCL-2 modulates mitochondrial respiration and mitochondrial ATP generation in addition to its established anti-apoptotic role. VEN disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitro and in vivo. Disclosures Konopleva: Ascentage: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Ablynx: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Kisoji: Consultancy; AstraZeneca: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding.

Pigment Dispersing Factors and Their Cognate Receptors in a Crustacean Model, With New Insights Into Distinct Neurons and Their Functions.

Pigment dispersing factors (PDFs, or PDHs in crustaceans) form a structurally related group of neuropeptides found throughout the Ecdysozoa and were first discovered as pigmentary effector hormones in crustaceans. In insects PDFs fulfill crucial neuromodulatory roles, most notably as output regulators of the circadian system, underscoring their central position in physiological and behavioral organization of arthropods. Intriguingly, decapod crustaceans express multiple isoforms of PDH originating from separate genes, yet their differential functions are still to be determined. Here, we functionally define two PDH receptors in the crab Carcinus maenas and show them to be selectively activated by four PDH isoforms: PDHR 43673 was activated by PDH-1 and PDH-2 at low nanomolar doses whilst PDHR 41189 was activated by PDH-3 and an extended 20 residue e-PDH. Detailed examination of the anatomical distribution of all four peptides and their cognate receptors indicate that they likely perform different functions as secreted hormones and/or neuromodulators, with PDH-1 and its receptor 43,673 implicated in an authentic hormonal axis. PDH-2, PDH-3, and e-PDH were limited to non-neurohemal interneuronal sites in the CNS; PDHR 41189 was largely restricted to the nervous system suggesting a neuromodulatory function. Notably PDH-3 and e-PDH were without chromatophore dispersing activity. This is the first report which functionally defines a PDHR in an endocrine system in a crustacean and to indicate this and other putative roles of this physiologically pivotal peptide group in these organisms. Thus, our findings present opportunities to further examine the endocrine and circadian machinery in this important arthropod phylum.

Ultra-low doses of the transient receptor potential vanilloid 1 agonist, resiniferatoxin, prevents vomiting evoked by diverse emetogens in the least shrew (Cryptotis parva).

Published studies have shown that the transient receptor potential vanilloid 1 (TRPV1) receptor agonist, resiniferatoxin (RTX), has pro and antiemetic effects. RTX can suppress vomiting evoked by a variety of nonselective emetogens such as copper sulfate and cisplatin in several vomit-competent species. In the least shrew, we have already demonstrated that combinations of ultra-low doses of RTX and low doses of the cannabinoid CB1/2 receptor agonist delta-9-tetrahydrocannabinol (Δ-THC) produce additive antiemetic effects against cisplatin-evoked vomiting. In the current study, we investigated the broad-spectrum antiemetic potential of very low nonemetic doses of RTX against a diverse group of specific emetogens including selective and nonselective agonists of serotonergic 5-hydroxytrptamine (5-HT3) receptor (5-HT and 2-Me-5-HT), dopaminergic D2 receptor (apomorphine and quinpirole), cholinergic M1 receptor (pilocarpine and McN-A-343), as well as the selective substance P neurokinin NK1 receptor agonist GR73632, the selective L-Type calcium channel agonist FPL64176, and the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. When administered subcutaneously, ultra-low (0.01 µg/kg) to low (5.0 µg/kg) doses of RTX suppressed vomiting induced by the aforementioned emetogens in a dose-dependent fashion with 50% inhibitory dose values ranging from 0.01 to 1.26 µg/kg. This study is the first to demonstrate that low nanomolar nonemetic doses of RTX have the capacity to completely abolish vomiting caused by diverse receptor specific emetogens in the least shrew model of emesis.

Inhibiting Mitochondria Function By Bcl-2 Inhibitor Venetoclax and Complex I Inhibitor Iacs-010759 Eliminate Leukemia Cells in Pre-Clinical AML Models

Previous studies have demonstrated that AML is BCL-2 dependent malignancy, and leukemia stem cell (LSC) rely on BCL-2 for survival (Pan, Cancer Discovery 2014;Lagadinou, Cell Stem Cell, 2013). Selective Bcl-2 inhibitor venetoclax (ABT-199) combined with azacitidine was reported to inhibit complex II of the mitochondrial transport chain in AML (Pollyea, Nat Med 2018). IACS-010759, a novel complex I inhibitor, demonstrated effective inhibition of cell respiration and potent anti-leukemia effect in AML pre-clinical models (Molina, Nat Med 2018). We designed the experiments to study the combined efficacy and mechanisms of action of venetoclax and IACS-010759 in AML. In vitro, priming of MOLM-13 cells with 20nM venetoclax for 24hrs followed by 10nM IACS-010759 for 1hr triggered 60% reduction in oxygen consumption rate (OCR), while only partial inhibition (<15% OCR reduction) was seen in cells treated with these agents separately. This translated in accelerated loss of the mitochondrial membrane potential by JC-1 flow cytometry, cleavage of caspase 3 and potent (>70%) reduction of viable cell numbers (OCI-AML2, MV-4-11, and MOLM-13). We have further shown by co-immunoprecipitation studies that venetoclax disrupts interaction of BCL-2 with the mitochondrial protein VDAC known to regulate ADP/ATP exchange during electron transport across mitochondria membrane. In addition, an MS-based metabolomics analysis indicated that ATP and CTP intracellular levels dropped to undetectable levels following treatment with ABT-199 (with or without IACS). ADP, GDP and UDP levels were unchanged with ABT-199; however, GDP levels dropped to undetectable levels following the combined treatment. Moreover, ABT-199 significantly increased intracellular levels of AMP, UMP, CMP and GMP and this accumulation of mono-nucleotides was enhanced by the combination of ABT-199 and IACS-010759. In primary AML samples (n=3) and PDX cells (n=4) cultured ex vivo, combined venetoclax and IACS-010759 at low nanomolar doses reduced viable cell numbers in an additive or synergistic fashion. To better understand the role of BCL-2 in cellular respiration, we examined the oxygen consumption rates (OCR) in control or Bcl-2-overexpressing HL-60 cells (a kind gift of Dr. K. Bhalla, MDACC). The HL-60/BCL-2 cells had higher basal and maximal OCR than the control cells by Seahorse analysis, and higher mitochondrial ROS production by H2DCFDA and MitoSOX Red flow cytometry. BCL-2 inhibition with 100nM venetoclax for 2 hrs induced ROS production in control HL-60 cells but not in cells with BCL-2 overexpression. Further, cells with BCL-2 overexpression were less sensitive to IACS-010759. These data suggest that BCL-2 facilitates cellular respiration and reduces efficacy of the mitochondrial inhibitors. Given recent accelerated FDA approval of venetoclax and azacitidine combination for elderly unfit AML, we next tested the efficacy of the "triple" combination of venetoclax, azacitidine and IACS-010759 in the in vivoAML PDX model. We injected AML PDX cells 3747422 harboring IDH1, NMP1, NRAS, CEBPA, FLT3-ITD mutations into NRG mice and upon engraftment, randomized mice into 4 groups to receive vehicle, venetoclax (50mg/kg, 5 days on/2 days off, day 1-21) with azacitidine (1.25mg/kg daily , day 1-7), IACS-010759 (1mg/kg, 5 days on/2 days off, day 1-14), or the triple combination. Therapy was well tolerated, without any apparent weight loss or toxicities. All therapies reduced circulating leukemia burden with the best efficacy seen in the triple-therapy cohort, with average circulating tumor burden of 31.2%, 6.9%, 5.1% and 0.4% in vehicle, IACS-010759, venetoclax/azacitidine and triple-therapy cohorts, respectively. Survival analysis and additional PDX models are ongoing and will be reported. In summary, these findings indicate that BCL-2 modulates mitochondrial respiration in addition to its established anti-apoptotic role. Venetoclax disrupts the BCL-2/VDAC interactions and reduces mitochondrial respiration, which is facilitated by the combined therapy with mitochondrial complex I inhibitor IACS-010759. Our preliminary findings indicate potent anti-AML activity of the dual and triple (with hypomethylating agent) combinations in vitroand in vivo. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; Calithera: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

Abstract 2711: Development of a new innovative multifunctional immune checkpoint inhibitor

Abstract Blockade of PD-1, PD-L1 or CTLA-4 is an attractive strategy for Immuno-Oncology (I-O) therapy. Moreover, combination approaches, which inhibit non-redundant pathways of PD-1/PD-L1 and CTLA-4, have been reported to show enhanced anti-tumor activity as well as improved progression-free survival in clinical trials of melanoma. The use of immune checkpoint blockade (ICB) drugs, however, can lead to severe or life-threatening complications of autoimmune disorders such as diabetes, thyroiditis, pulmonary fibrosis and vitiligo. Therefore, new innovations are critical to overcome possibilities of unintended immunoactivation and minimize drug cross-reactions and complications. Here, we designed and synthesized a novel alkylating Pyrrole-Imidazole (PI) polyamide with indole-seco-CBI capable of disrupting expressions of human PD-1, PD-L1 and CTLA-4 genes (CCC07-01) by binding to a common motif. Our initial evaluation process confirmed that the induction of tumor- and tumor-environment-specific combinatorial ICB therapy was possible by only a single agent. CCC07-01 administration significantly suppressed PD-1, PD-L1 and CTLA-4 mRNA and protein expressions at low nanomolar doses (15, 10, 5 nM respectively) in several cell lines. Furthermore, logP characterization experiments found CCC07-01 to be sufficiently lipophilic that enhanced the molecule's capability to accumulate and be retained in tumor tissues. Additionally, elevated doses of CCC07-01 induced substantial cytotoxicity in SW480, RKO and A2058 cancer cells (IC50=27, 12 and 11 nM, respectively) but primary cells derived from normal human tissues were not affected. These results illuminated the therapeutic possibility of CCC07-01 through, at low doses, its anti-tumor effect in tumor microenvironments and generalized cytotoxicity at increased dosage levels. CCC07-01's enhanced retention in tumor may also contribute to lower risks of triggering adverse autoimmune events compared to other ICB approaches. Preliminary results from human RKO colon carcinoma xenograft NGS mice, following the engraftment of human HLA-matched peripheral blood mononuclear cells (PBMC), showed significant tumor reduction 14 days after the injection of CCC07-01 (2.3 mg/kg, p < 0.05) and displayed detectable amounts of predominant effector memory CD8 T cells in the spleen 14 days post-administration (9.2 mg/kg, p < 0.05). The approach of simultaneously targeting elements in immune checkpoints, as demonstrated by CCC07-01, can lead to new therapeutic opportunities and reduce healthcare costs by curbing undesired autoimmune complications. Together with Zenyaku Kogyo, we envision a breakthrough in the field of current antibody-based ICB therapy as we continue to evolve and improve the use of PI polyamides as a viable candidate for ICB. Citation Format: Hiroki Nagase, Keiko Fukushima, Asuka Hattori, Mayu Shinohara, Atsushi Takatori, Takayoshi Watanabe, Nobuko Koshikawa, Takahiro Inoue, Jason Lin, Yoshinao Shinozaki. Development of a new innovative multifunctional immune checkpoint inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2711.

Using embryonic zebrafish to evaluate the effects of exposure to novel bisphenol analogues

Bisphenol A (BPA) is a well‐established endocrine disruptor that has been shown to induce developmental and behavioral changes in both human and animal models. Gestational exposure of embryonic zebrafish to low nanomolar doses of bisphenol A (or S) altered hypothalamic neurogenesis during development and induced hyperactive swimming behaviors. There are many potential bisphenol analogues, but little is known about their toxicity or ability to act as endocrine disruptors. We have synthesized a small library of novel bisphenol analogues and are testing their toxicity in a zebrafish larval toxicity assay. We are also using an ELISA‐based estrogen binding assay to assess the analogues' affinity for estrogen receptors. We will be exposing zebrafish embryos to ranging concentrations of bisphenol analogues during time periods of 10–16 hpf (hours post‐fertilization), 16–24 hpf, and 24–36 hpf. At 5 dpf (days post‐fertilization) zebrafish locomotor activity in control and analogue exposed fish will be assessed using video‐tracking software quantifying bursts of hyperactivity. Results from these experiments will be discussed.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P1-10-03: Precision therapeutic combinations are synergistic against triple negative breast cancer using compensatory pathways

Abstract Introduction: Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer cases in the United States, and despite its lower incidence, contributes to a disproportionately higher rate of morbidity and mortality compared to other breast cancer subtypes. In an effort to treat TNBC, a cancer that has no targeted therapies, many have chosen to experiment with combinations of drugs that are chosen for their genomic expression or “hard targets”. It has been previously noted however, that single agent therapeutics can change the genomic landscape of both mice and human cells and may be responsible for resistance. Here we show that targeting a compensatory pathway after treatment with a single therapy, results in synergistic combinations and can outperform the choice of hard target therapies. Methods: Eight TNBC cell lines were chosen based on their abundance of clinically actionable targets. The primary hard target combinations were chosen using data from TCGA and a board consisting of oncologists and researchers at Indiana University School of Medicine. Compensatory therapies were found using RNA sequencing data from untreated versus single therapeutic treated TNBC cell lines. The merged transcript RPKMs were transformed and analyzed for differential expression. Statistically significant genes were imported into Ingenuity Pathway Analysis (IPA) to identify either therapeutics or genomic targets using the Causal network analysis and Upstream regulator functions. All drug combinations were tested in their respective cell lines and cell viability was assessed via Celltiter-Fluor. Synergy of the combinations was calculated using the Chou-Talalay method. Results: Using genomically chosen hard targets for drug combinations, all eight cell lines displayed additive or antagonistic results except low nanomolar doses for the MDA-MB-231 cell line. Dosing of MDA-MB-231s with Debrafinib and Pazopanib however, turned highly antagonistic as dosing increased. Using next-generation RNA sequencing data of TNBCs, IPA analysis identified several compensatory targets for each cell line when treated with one of the primary genomically driven drug at its IC50. Using dose escalation of the new drugs with a single hard target drug, we found that each compensatory combination displayed a striking increase in synergy across all TNBC cell lines treated when compared to their original hard target combination. Conclusion: RNA sequencing of TNBC cells lines treated with single therapies chosen by actionable genomic landscape has revealed compensatory pathways, indicating further druggable targets. These compensatory pathways have been observed to more efficacious in treating TNBC cell lines. Using therapeutics that are either FDA approved or in clinical trials we have found that each compensatory combination shows a higher level of synergy across all cell lines. These data show that choosing a secondary therapy based on compensatory pathways may outperform hard target combinations in the clinic. Citation Format: Solzak J, Hancock B, Paul R, Radovich M. Precision therapeutic combinations are synergistic against triple negative breast cancer using compensatory pathways [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-03.

Synergistic effect of a novel autophagy inhibitor and Quizartinib enhances cancer cell death

Drug combinations have been increasingly applied in chemotherapy as a strategy to enhance the efficacy of anti-cancer treatment. The appropriate drug combinations may achieve synergistic effects beyond monotherapies alone. AC220 (Quizartinib), an FLT3 receptor tyrosine kinase inhibitor, developed for the treatment of AML, has been tested in phase II human clinical trials. However, AC220 as a monotherapy is not efficacious enough. In this study, we performed a small-molecule screening of 12 640 compounds in order to find a compound that increase the AC220 efficacy in chemotherapy. We identified that TAK-165, a HER2 inhibitor, even when used at low nanomolar doses in combination with AC220, was able to induce cell death in different cancer cells, but not in non-cancer cell lines. We showed that TAK-165 and AC220 act synergistically to downregulate key signaling pathways and potently induce cancer cell death. Furthermore, we demonstrated that TAK-165 inhibited autophagy in a HER2-independent manner. Finally, we showed that the combination of TAK-165 and AC220 induced cell death in cancer cells through the activation of chaperone-mediated autophagy. Overall, these findings support the strategy for using AC220 and an autophagy inhibitor such as TAK-165 in a combinatorial treatment to enhance the efficacy of cancer therapies.

Abstract 5163: Characterization of NMS-E194, a selective and potent PERK inhibitor with efficacy in the KMS-11 multiple myeloma

Abstract PERK (PKR-like endoplasmic reticulum kinase) is a serine-threonine kinase associated to endoplasmic reticulum membrane. Together with ATF6 and IRE1, PERK is a key effector of the Unfolded Protein Response (UPR), a network of signaling pathways that ensures protein homeostasis in the endoplasmic reticulum. Multiple Myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, and inhibition of PERK may be an effective strategy to inhibit growth of these tumors. Here we describe the in vitro and in vivo properties of NMS-E194, a novel potent and selective ATP-competitive PERK inhibitor belonging to the arylsulfonamide chemical class. NMS-E194 inhibits PERK kinase with a Ki of ca 1.5 nM in biochemical assay and possesses high selectivity towards a panel of 54 kinases. When tested in cell proliferation assays, NMS-E194 is preferentially active on multiple myeloma (MM) and diffuse large B cell lymphoma cell lines. In mechanism of action studies, NMS-E194 shows a bi-phasic behaviour: at low nanomolar doses it activates PERK, causing ATF4 accumulation, while at >200 nM it inhibits PERK and downstream pathway, inducing apoptosis. NMS-E194 possesses a favourable in vitro ADME profile and has promising PK properties in the mouse. NMS-E194 demonstrated strong anti-tumor activity following oral administration at 25 mg/kg daily to mice harbouring luciferized KMS-11 cells in a disseminated growth model of MM, with no overt toxicity. Overall, the data available so far warrant further development of NMS-E194 and support PERK inhibition as a novel therapeutic approach in MM and other PERK-dependent tumors. Citation Format: Claudia Perrera, Maurizio Pulici, Davide Carenzi, Ilaria Motto, Marina Fasolini, Elena Casale, Stefania Re Depaolini, Simona Rizzi, Daniela Asa, Fulvia Roletto, Simona Bindi, Daniele Casero, Patrizia Banfi, Elena Ardini, Nadia Amboldi, Dario Ballinari, Fabio Gasparri, Sabrina Cribioli, Laura Mancini, Marina Ciomei, Daniele Donati, Eduard Felder, Arturo Galvani, Antonella Isacchi, Barbara Valsasina. Characterization of NMS-E194, a selective and potent PERK inhibitor with efficacy in the KMS-11 multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5163. doi:10.1158/1538-7445.AM2017-5163

Abstract 1002: An epigenetic strategy to degrade the estrogen receptor in breast cancer

Abstract Epigenetic changes in DNA methylation can lead to altered gene expression and the development and progression of breast cancer. Luminal breast tumors are associated with increased DNA hypermethylation of CpG islands while basal, triple negative tumors more commonly have lower levels of DNA methylation. Increasing evidence suggests that aberrant epigenetic alterations in DNA methylation and chromatin structure may also contribute to endocrine resistance in luminal breast cancers. We thus asked whether epigenetic therapy using the demethylating agent, 5-Azacytidine (AZA) is an effective anti-tumorigenic agent in luminal breast cancers, what epigenetic mechanisms might be involved in the response and whether AZA could play a role in the sensitization of tamoxifen resistant cells. We have demonstrated using 26 breast cancer cell lines that luminal B, ER+ breast cancer is more responsive to treatment with AZA than are Her2+ or triple negative (TN) breast cancers. Cells were treated in culture with low nanomolar doses of AZA for 72 hrs, followed by transplantation into mice and weekly palpation of tumors. Tumorgraft measurements show that 67% of luminal B, ER+ cells are sensitive to AZA, whereas only 22% of TN and 25% of Her2+ cells exhibit an anti-tumorigenic response to AZA in mice. Expression data showed that AZA leads to decreases in ER mRNA and protein expression in 50% and 67% of luminal B lines respectively. Further investigation showed that a longer AZA treatment led to lower ERα protein levels and stronger tumor growth inhibition. Protease inhibitor (MG132) treatment rescued ERα protein reduction in 50% of the luminal B cell lines, suggesting that an AZA-mediated, ubiquitin-26S proteasomal pathway may be responsible for the degradation of ER. Gene expression analysis demonstrated that the E1 ubiquitin-activating enzyme, (UBA7-UBE1L) was up-regulated at the mRNA and protein levels by AZA. UBA7 has been suggested to function as a tumor suppressor in lung cancer and elevations in UBA7 have been correlated with longer overall survival in breast cancer. The interaction of UBA7 with ERα protein was validated by immunoprecipitation and over-expression of UBA7 in ZR-75-1 cells led to a reduction in ERα protein. We further analyzed the UBA7 promoter region by Illumina 450K array and bisulfite sequencing in our cell lines and identified two CpG rich sequences in the promoter that may be important for AZA induced UBA7 promoter demethylation and gene re-expression. We also demonstrated in both tamoxifen sensitive and resistant cells that combination therapy with both AZA and tamoxifen was more effective at inhibiting tumor growth than the single agents. Studies are ongoing in our lab to better understand this response. Our data suggest that AZA may be clinically useful to inhibit the growth of some luminal breast tumors via its actions to increase expression of UBA7 and to degrade ER and may have efficacy in combination with anti-estrogen treatments. Citation Format: Huili Li, Foteinos-Ioannis Dimitrakopoulos, Meredith Stone, Lauren Murphy, Stephen Baylin, Cynthia A. Zahnow. An epigenetic strategy to degrade the estrogen receptor in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1002. doi:10.1158/1538-7445.AM2017-1002

6α-Acetoxyanopterine: A Novel Structure Class of Mitotic Inhibitor Disrupting Microtubule Dynamics in Prostate Cancer Cells.

The lack of a cure for metastatic castrate-resistant prostate cancer (mCRPC) highlights the urgent need for more efficient drugs to fight this disease. Here, we report the mechanism of action of the natural product 6α-acetoxyanopterine (6-AA) in prostate cancer cells. At low nanomolar doses, this potent cytotoxic alkaloid from the Australian endemic tree Anopterus macleayanus induced a strong accumulation of LNCaP and PC-3 (prostate cancer) cells as well as HeLa (cervical cancer) cells in mitosis, severe mitotic spindle defects, and asymmetric cell divisions, ultimately leading to mitotic catastrophe accompanied by cell death through apoptosis. DNA microarray of 6-AA-treated LNCaP cells combined with pathway analysis identified very similar transcriptional changes when compared with the anticancer drug vinblastine, which included pathways involved in mitosis, microtubule spindle organization, and microtubule binding. Like vinblastine, 6-AA inhibited microtubule polymerization in a cell-free system and reduced cellular microtubule polymer mass. Yet, microtubule alterations that are associated with resistance to microtubule-destabilizing drugs like vinca alkaloids (vinblastine/vincristine) or 2-methoxyestradiol did not confer resistance to 6-AA, suggesting a different mechanism of microtubule interaction. 6-AA is a first-in-class microtubule inhibitor that features the unique anopterine scaffold. This study provides a strong rationale to further develop this novel structure class of microtubule inhibitor for the treatment of malignant disease. Mol Cancer Ther; 16(1); 3-15. ©2016 AACR.

Abstract LB-158: Simultaneous targeting of the cell cycle and transcription through irreversible inhibition of CDK7: a novel, new therapeutic approach for high-grade glioma

Abstract High Grade Glioma (HGG), an incurable aggressive neoplasm of the brain, is characterised by a diverse array of genetic aberrations including those associated with receptor tyrosine kinase (RTK) signalling and cell cycle regulation pathways. Furthermore, HGG possess a highly heterogeneous intratumoral transcriptional profile, both at the global and subclonal levels, that allows for rapid resistance to a wide spectrum of therapeutics. Cyclin dependent kinase 7 (CDK7) presents an opportunity to target proliferation and global transcription simultaneously as it is the CDK activating kinase that controls multiple checkpoint progressions through the cell cycle as well as stabilising the RNA polymerase II-based transcriptional apparatus. Using a new irreversible inhibitor of CDK7, THZ1, we demonstrate that a panel of 10 patient-derived primary HGG cells lines, representing a diverse array of genotypes and phenotypes, were all exquisitely sensitive to THZ1 treatment at low nanomolar doses. THZ1 induced global transcription downregulation as measured by microarray analysis and resulted in potent caspase 3/7 activation, DNA damage and apoptosis. Cell cycle in all lines was arrested at the G2/M phase regardless of p53, CDKN2A/B or RB status, with substantial aneuploidy observed. Additionally, THZ1 treatment results in the dramatic loss of several RTKs critical in driving HGG including EGFR, EGFRvIII, PDGFR-α, PDGFR-β, MET, FGFR-3 and AXL, resulting in reduced PI3K/AKT, MAPK/ERK and STAT3 activation. Efficacy in ex vivo tumor slices and in vivo orthotopic experiments will be presented as well as insights into possible mechanisms of resistance. Our work suggest that irreversible inhibition of CDK7 represents a novel and effective treatment for HGG. Citation Format: Sameer Greenall, Terrance G. Johns. Simultaneous targeting of the cell cycle and transcription through irreversible inhibition of CDK7: a novel, new therapeutic approach for high-grade glioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-158.