Dysregulation and Epigenetic Reprogramming of NRF2 Signaling Axis Promote Acquisition of Cisplatin Resistance and Metastasis in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. EXPERIMENTAL DESIGN: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. RESULTS: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CONCLUSIONS: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

In vitro expansion and differentiation of fresh and revitalized adult canine bone marrow-derived and adipose tissue-derived stromal cells.

The objective of this study was to determine the tissue density, in vitro expansion and differentiation of canine adipose tissue-derived (ASC) and bone marrow-derived (BMSC) stromal cells. Primary (P0) and cell passages 1-6 (P1-6) cell doubling numbers (CD) and doubling times (DT) were determined in fresh cells. The P0, P3, and P6 adipogenic (CFU-Ad), osteogenic (CFU-Ob), and fibroblastic (CFU-F) colony forming unit frequencies, lineage specific mRNA levels in differentiated P3 cells and composition of P3 and P6 chondrogenic pellets were assessed in cryogenically preserved cells. Cell yields from bone marrow were significantly higher than adipose tissue. Overall ASC and BMSC CDs and DTs and P3 and P6 CFU-F, CFU-Ad, and CFU-Ob were comparable. The P0 BMSC CFU-Ob was significantly higher than ASC. Lineage specific mRNA levels were higher in differentiated versus control cells, but similar between cell types. Protein was significantly greater in P3 versus P6 ASC chondrogenic pellets. Based on these findings, fresh and revitalized canine ASCs are viable alternatives to BMSCs for stromal cell applications.

Impaired expansion and multipotentiality of adult stromal cells in a rat chronic alcohol abuse model.

It is well established that bone maintenance and healing is compromised in alcoholics. Adult bone marrow-derived stromal cells (BMSCs) and adipose tissue-derived stromal cells (ASCs) likely contribute to bone homeostasis and formation. Direct and indirect alcohol exposure inhibits osteoprogenitor cell function through a variety of proposed mechanisms. The goal of this study was to characterize the effects of chronic alcohol ingestion on the native number and in vitro growth characteristics and multipotentiality of adult BMSCs and ASCs in a rat model. Adult male Sprague-Dawley rats received a liquid diet containing 36% ethanol or an isocaloric substitution of dextramaltose (control). After 4, 8, or 12 weeks of the diet, ASCs were harvested from epididymal adipose tissue and BMSCs from femoral and tibial bone marrow. Cell doublings (CDs) per day and doubling times (DTs) were determined for primary cells (P0) and cell passages 1 through 6 (P1-P6). Fibroblastic (CFU-F), adipogenic (CFU-Ad), and osteogenic (CFU-Ob) colony-forming unit (CFU) frequencies were assessed for P0, P3, and P6. The CDs and DTs were lower and higher, respectively, for ASCs and BMSCs harvested from ethanol versus control rats at all time points. The CFU-F, CFU-Ad, and CFU-Ob were significantly higher in ASCs harvested from control versus ethanol rats for P0, P3, and P6 at all times. Both CFU-Ad and CFU-Ob were significantly higher in P0 BMSCs harvested from control versus ethanol rats after 12 weeks of the diet. The CFU-Ob for P3 BMSCs from control rats was significantly higher than those from ethanol rats after 8 and 12 weeks on the diet. All three CFU frequencies in ASCs from ethanol rats tended to decrease with increasing diet duration. The ASC cell and colony morphology was different between control and ethanol cohorts in culture. These results emphasize the significant detrimental effects of chronic alcohol ingestion on the in vitro expansion and multipotentiality of adult mesenchymal stromal cells (MSCs). Maintenance of the effects through multiple cell passages in vitro suggests cells may be permanently compromised.

Mesenchymal stromal cells: past, present, and future.

Adult mesenchymal stromal cells are plastic-adherent cells that are self-renewing and have the capacity to differentiate into various tissue specific lineages. Stromal cells were initially discovered over 100 years ago and substantial insight into stromal cell identification, isolation, characterization, and differentiation has been made, including efforts to elucidate the factors involved in stromal cell differentiation. Stromal cells have immune privilege and thus are attractive candidates for tissue engineering and regenerative medicine applications. Positive results from a number of recent investigations support the use of adult mesenchymal stromal cells for clinical application. This review article provides a brief overview of past, present, and future stromal cell technology.

Immunogenicity of allogeneic adipose-derived stem cells in a rat spinal fusion model.

Adipose-derived stem cells (ASCs) express a nonimmunogenic profile as shown by in vitro studies that demonstrate a lack of T cell proliferation to allogeneic ASCs as well as ASC-mediated suppression of mixed lymphocyte reactions. To determine whether these observations would translate in vivo, immune monitoring studies were carried out in conjunction with a rat spinal fusion study. ASCs derived from Fischer or ACI strain rats were loaded onto scaffolds and implanted in Fischer recipients that had undergone the following treatments: (1) No treatment; (2) Scaffold only; (3) Syngeneic ASCs+Scaffold; or (4) Allogeneic ASCs+Scaffold. Half of each group was sacrificed at 4 weeks postimplantation, and the remaining animals were sacrificed at 8 weeks. As determined in a separate study, allogeneic and syngeneic ASCs were equally efficacious in accelerating spinal fusion compared to No treatment and Scaffold only control groups. To determine whether donor ASCs induced an immune response in recipient rats, lymph nodes were harvested for T cell proliferation studies and serum was collected to assess antibody responses. Although T cell priming was not detected to donor alloantigens in recipients at either time point, significant antibody responses were detected to ACI ASCs in animals implanted with syngeneic or allogeneic ASCs. Antibodies were of the IgG isotype, noncytotoxic in the presence of complement, and reactive to fetal bovine serum. These results support the use of allogeneic ASCs for spinal fusion.

Acceleration of spinal fusion using syngeneic and allogeneic adult adipose derived stem cells in a rat model.

Posterolateral spinal fusion is the standard treatment for lumbar compression fractures. Adult adipose tissue-derived stem cells (ASCs) promote osteogenesis in vivo and in vitro. The hypothesis tested in this study was that syngeneic and allogeneic ASCs on a biomaterial scaffold composed of tricalcium phosphate and collagen I will accelerate spinal fusion in a rat model. ASCs from male Fischer or ACI rats were loaded onto scaffolds (53,571 cells/mm(3)) and cultured in stromal media for 48 h. Male Fisher rats were assigned to 4 cohorts (n = 14/cohort) after bilateral decortication of the L4 and L5 transverse processes: (1) No treatment; (2) scaffold only; (3) scaffold + syngeneic ASCs; or (4) scaffold + allogeneic ASCs. Half of each cohort was harvested 4 or 8 weeks after surgery. Spinal fusion was evaluated with radiographs, microcomputed tomography, and light microscopy. Callus did not form in spines without scaffolds. There were no significant differences in callus formation among scaffold cohorts 4 weeks after surgery. Callus formation was more mature in both ASC cohorts versus scaffold alone 8 weeks after surgery based on microstructure as well as radiographic and microcomputed tomographic evidence of active bone formation. Inflammatory cell infiltrate was significantly lower in both ASC cohorts (syngeneic = 18.3 +/- 0.85%; allogeneic = 23.5 +/- 2.33%) versus scaffold alone (46.8 +/- 11.8%) 4 weeks after surgery. Results of this study support syngeneic and allogeneic ASC acceleration of posterior lumbar spinal fusion in a rat model.