Search for Synergistic Drug Combinations to Treat Chronic Lymphocytic Leukemia.

Finding synergistic drug combinations is an important area of cancer research. Here, we sought to rationally design synergistic drug combinations with an inhibitor of BTK kinase, ibrutinib, which is used for the treatment of several types of leukemia. We (a) used a pooled shRNA screen to identify genes that protect cells from the drug, (b) identified protective pathways via bioinformatics analysis of these gene sets, and (c) identified drugs that inhibit these pathways. Based on this analysis, we established that inhibitors of proteasome and mTORC1 could synergize with ibrutinib both in vitro and in vivo. We suggest that FDA-approved inhibitors of these pathways could be effectively combined with ibrutinib for the treatment of chronic lymphocytic leukemia (CLL).

Hsp70-Bag3 Module Regulates Macrophage Motility and Tumor Infiltration via Transcription Factor LITAF and CSF1.

The molecular chaperone Hsp70 has been implicated in multiple stages of cancer development. In these processes, a co-chaperone Bag3 links Hsp70 with signaling pathways that control cancer development. Recently, we showed that besides affecting cancer cells, Hsp70 can also regulate the motility of macrophages and their tumor infiltration. However, the mechanisms of these effects have not been explored. Here, we demonstrated that the Hsp70-bound co-chaperone Bag3 associates with a transcription factor LITAF that can regulate the expression of inflammatory cytokines and chemokines in macrophages. Via this interaction, the Hsp70-Bag3 complex regulates expression levels of LITAF by controlling its proteasome-dependent and chaperone-mediated autophagy-dependent degradation. In turn, LITAF regulates the expression of the major chemokine CSF1, and adding this chemokine to the culture medium reversed the effects of Bag3 or LITAF silencing on the macrophage motility. Together, these findings uncover the Hsp70-Bag3-LITAF-CSF1 pathway that controls macrophage motility and tumor infiltration.

Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2α via the Hsp70-Bag3 module.

The major heat shock protein Hsp70 forms a complex with a scaffold protein Bag3 that links it to components of signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, to search for pathways regulated by the complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. RNAseq followed by the pathway analysis indicated that several signaling pathways including UPR were activated by JG-98. Surprisingly, only the eIF2α-associated branch of the UPR was activated, while other UPR branches were not induced, suggesting that the response was unrelated to the ER proteotoxicity and ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was driven by a distinct eIF2α kinase HRI. Hsp70-Bag3 directly interacted with HRI and regulated eIF2α phosphorylation upon cytoplasmic proteotoxicity. Therefore, cytosolic proteotoxicity can activate certain UPR genes via Hsp70-Bag3-HRI-eIF2α axis.

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects.

Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall, a cell double-barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies.

A novel, dual action chimera comprising DNA methylating agent and near-IR xanthene-cyanine photosensitizer for combined anticancer therapy.

A facile synthesis, biological evaluation and photodynamic properties of novel activatable anticancer molecular hybrids (chimeras) Ch and I-Ch are described. The chimeras consist of DNA methylating methyl triazene moiety and fluorogenic xanthene-cyanine (XCy) or iodinated xanthene-cyanine (I-XCy) photosensitizer. These two anticancer core structures are bound by means of a self-immolative 4-aminobenzyl alcohol linker. The hydrolytic cleavage of the carbamate protecting group promotes activation of both DNA methylating monomethyl triazene and phototoxic xanthene-cyanine dye providing, in addition, a near-IR emission signal for detection of the drug activation events. Preliminary antiproliferative assay demonstrates that the developed chimeras exhibit higher antitumor activity in the breast cancer cell line upon near-IR light irradiation compared to their structural constituents, xanthene-cyanine photosensitizer and monomethyl triazene substance.

A Transgenic Model Reveals the Role of Klotho in Pancreatic Cancer Development and Paves the Way for New Klotho-Based Therapy.

Klotho is an anti-aging transmembrane protein, which can be shed and can function as a hormone. Accumulating data indicate that klotho is a tumor suppressor in a wide array of malignancies, and designate the subdomain KL1 as the active region of the protein towards this activity. We aimed to study the role of klotho as a tumor suppressor in pancreatic ductal adenocarcinoma (PDAC). Bioinformatics analyses of The Cancer Genome Atlas (TCGA) datasets revealed a correlation between the survival of PDAC patients, levels of klotho expression, and DNA methylation, and demonstrated a unique hypermethylation pattern of klotho in pancreatic tumors. The in vivo effects of klotho and KL1 were examined using three mouse models. Employing a novel genetic model, combining pancreatic klotho knockdown with a mutation in Kras, the lack of klotho contributed to PDAC generation and decreased mousece survival. In a xenograft model, administration of viral particles carrying sKL, a spliced klotho isoform containing the KL1 domain, inhibited pancreatic tumors. Lastly, treatment with soluble sKL prolonged survival of Pdx1-Cre; KrasG12D/+;Trp53R172H/+ (KPC) mice, a model known to recapitulate human PDAC. In conclusion, this study provides evidence that klotho is a tumor suppressor in PDAC. Furthermore, these data suggest that the levels of klotho expression and DNA methylation could have prognostic value in PDAC patients, and that administration of exogenous sKL may serve as a novel therapeutic strategy to treat PDAC.

Discovery of Dolastatinol: A Synthetic Analog of Dolastatin 10 and Low Nanomolar Inhibitor of Tubulin Polymerization.

We developed a highly potent anticancer agent, dolastatinol, which is a methylene hydroxyl derivative of dolastatin 10. Dolastatinol is a synthetic analog of dolastatin 10, synthesized by a solid-phase peptide Fmoc chemistry protocol on 2-chlorotrityl chloride resin utilizing a pH-triggering self-immolative monosuccinate linker. The introduction of the C-terminus hydroxyl methylene functionality preserves the anticancer properties of the parent dolastatin 10, including strong suppression of the cell proliferation, migration, high cytotoxicity. Our research establishes a new facile route toward the further development of C-terminus-modified dolastatin-10-based microtubule inhibitors for anticancer treatment.

Ratiometric Fluorescence Monitoring of Antibody-Guided Drug Delivery to Cancer Cells.

Ratiometric measurements utilizing two independent fluorescence signals from a dual-dye molecular system help to improve the detection sensitivity and quantification of many analytical, bioanalytical, and pharmaceutical assays, including drug delivery monitoring. Nevertheless, these dual-dye conjugates have never been utilized for ratiometric monitoring of antibody (Ab)-guided targeted drug delivery (TDD). Here, we report for the first time on the new, dual-dye TDD system, Cy5s-Ab-Flu-Aza, comprising the switchable fluorescein-based dye (Flu) linked to the anticancer drug azatoxin (Aza), reference pentamethine cyanine dye (Cy5s), and Her2-specific humanized monoclonal Trastuzumab (Herceptin) antibody. The ability of ratiometric fluorescence monitoring of drug release was demonstrated with this model system in vitro in the example of the human breast cancer SKBR3 cell line overexpressing Her2 receptors. The proposed approach for designing ratiometric, antibody-guided TDD systems, where a "drug-switchable dye" conjugate and a reference dye are independently linked to an antibody, can be expanded to other drugs, dyes, and antibodies. Replacement of the green-emitting dye Flu, which was found not detectable in vivo, with a longer-wavelength (red or near-IR) switchable fluorophore should enable quantification of drug release in the body.