Cell Adhesion-mediated Drug Resistance Research Articles

Abstract 4618: PIM kinases drive cell adhesion mediated drug resistance in prostate cancer

Abstract Introduction: Castration-resistant prostate cancer (CRPC) is an incurable and lethal progression of the disease that does not respond to any treatment options. Cell adhesion to the extracellular matrix (ECM) activates survival pathways, allowing cancer cells to evade anti-cancer therapy, a phenomenon known as cell adhesion-mediated drug resistance. Bone, which is the primary site of prostate cancer metastasis, is known to be enriched in laminin, a ubiquitous ECM protein. In addition, laminin binding integrins, α6β1 and α3β1, are the major integrins expressed in CRPC, suggesting that laminin-dependent adhesion provides a survival advantage. Based on these findings, activation of the laminin-binding integrin, α6β1, is an intriguing factor mediating resistance to therapy in CRPC. PIM1 is an oncogenic Ser/Thr kinase that is elevated in CRPC and is known to promote resistance to therapy. In contrast, to many pro-survival kinases, PIM kinases do not possess any obvious regulatory domains. Current evidence suggests that PIM1 is constitutively active when it is expressed, so PIM1 expression level directly correlates with its catalytic activity. Despite this fact, little is known about the internal and external mechanisms that dictate PIM1 protein levels and spatial activation in cancer cells. Here, we identify PIM1 as a key survival signal that contributes to cell adhesion-mediated drug resistance. Experimental procedures: Supporting the importance of this signaling axis in human tumors, multiplex IHC of a TMA containing prostate adenocarcinoma and CRPC bone metastases to show that integrin α6 and PIM1 are coexpressed and spatially correlated in primary and metastatic prostate cancer. To examine the spatiotemporal dynamics of PIM1 during cell adhesion, We developed a kinase activity reporter (PIMKAR) to monitor PIM catalytic activity in live cells. Because we observed PIM was highly localized to the membrane in patient tumors, we tagged PIMKAR with a myristylation sequence to specifically measure PIM activity at the membrane vs cytosol. Immunofluorescence and biochemical techniques were used to demonstrate the effect of PIM1 on mitophagy and oxidative stress. Conclusions: We demonstrate that activation of integrin α6β1 downstream of cell adhesion stabilizes PIM1 by blocking its proteasomal degradation. The resulting upregulation of PIM1 serves to reduce oxidative stress through complementary mechanisms reducing mitochondrial fragmentation and increasing the removal of damaged mitochondria by increasing mitophagy. Mechanistically, PIM1 upregulates BNIP3 to enhance mitophagy and thereby reduce oxidative stress in the cell. Together, these results expand our understanding of the mechanisms regulating PIM1 activation and provide evidence for the use of PIM inhibitors in bone mCRPC and other cancers where laminin is a major component of the ECM Citation Format: Caitlyn Flores, Shailender Chauhan, Cynthia Miranti, Anne Cress, Gregory Rogers, Noel Warfel. PIM kinases drive cell adhesion mediated drug resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4618.

Development of a cell adhesion-based prognostic model for multiple myeloma: Insights into chemotherapy response and potential reversal of adhesion effects.

Multiple myeloma (MM) is a hematologic malignancy notorious for its high relapse rate and development of drug resistance, in which cell adhesion-mediated drug resistance plays a critical role. This study integrated four RNA sequencing datasets (CoMMpass, GSE136337, GSE9782, and GSE2658) and focused on analyzing 1706 adhesion-related genes. Rigorous univariate Cox regression analysis identified 18 key prognosis-related genes, including KIF14, TROAP, FLNA, MSN, LGALS1, PECAM1, and ALCAM, which demonstrated the strongest associations with poor overall survival (OS) in MM patients. To comprehensively evaluate the impact of cell adhesion on MM prognosis, an adhesion-related risk score (ARRS) model was constructed using Lasso Cox regression analysis. The ARRS model emerged as an independent prognostic factor for predicting OS. Furthermore, our findings revealed that a heightened cell adhesion effect correlated with tumor resistance to DNA-damaging drugs, protein kinase inhibitors, and drugs targeting the PI3K/Akt/mTOR signaling pathway. Nevertheless, we identified promising drug candidates, such as tirofiban, pirenzepine, erlotinib, and bosutinib, which exhibit potential in reversing this resistance. In vitro, experiments employing NCIH929, RPMI8226, and AMO1 cell lines confirmed that MM cell lines with high ARRS exhibited poor sensitivity to the aforementioned candidate drugs. By employing siRNA-mediated knockdown of the key ARRS model gene KIF14, we observed suppressed proliferation of NCIH929 cells, along with decreased adhesion to BMSCs and fibronectin. This study presents compelling evidence establishing cell adhesion as a significant prognostic factor in MM. Additionally, potential molecular mechanisms underlying adhesion-related resistance are proposed, along with viable strategies to overcome such resistance. These findings provide a solid scientific foundation for facilitating clinically stratified treatment of MM.

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

The Role of the Microenvironment and Cell Adhesion Molecules in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy whose progression largely depends on the lymph node and bone marrow microenvironment. Indeed, CLL cells actively proliferate in specific regions of these anatomical compartments, known as proliferation centers, while being quiescent in the blood stream. Hence, CLL cell adhesion and migration into these protective niches are critical for CLL pathophysiology. CLL cells are lodged in their microenvironment through a series of molecular interactions that are mediated by cellular adhesion molecules and their counter receptors. The importance of these adhesion molecules in the clinic is demonstrated by the correlation between the expression levels of some of them, in particular CD49d, and the prognostic likelihood. Furthermore, novel therapeutic agents, such as ibrutinib, impair the functions of these adhesion molecules, leading to an egress of CLL cells from the lymph nodes and bone marrow into the circulation together with an inhibition of homing into these survival niches, thereby preventing disease progression. Several adhesion molecules have been shown to participate in CLL adhesion and migration. Their importance also stems from the observation that they are involved in promoting, directly or indirectly, survival signals that sustain CLL proliferation and limit the efficacy of standard and novel chemotherapeutic drugs, a process known as cell adhesion-mediated drug resistance. In this respect, many studies have elucidated the molecular mechanisms underlying cell adhesion-mediated drug resistance, which have highlighted different signaling pathways that may represent potential therapeutic targets. Here, we review the role of the microenvironment and the adhesion molecules that have been shown to be important in CLL and their impact on transendothelial migration and cell-mediated drug resistance. We also discuss how novel therapeutic compounds modulate the function of this important class of molecules.

Unlocking Drug Resistance in Multiple Myeloma: Adipocytes as Modulators of Treatment Response.

Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal proliferation of malignant plasma cells. Despite the development of a diverse array of targeted drug therapies over the last decade, patients often relapse and develop refractory disease due to multidrug resistance. Obesity is a growing public health threat and a risk factor for multiple myeloma, although the mechanisms by which obesity contributes to MM growth and progression have not been fully elucidated. In the present study, we evaluated whether crosstalk between adipocytes and MM cells promoted drug resistance and whether this was amplified by obesity. Human adipose-derived stem cells (ASCs) from nineteen normal (BMI = 20-25 kg/m2), overweight (25-30 kg/m2), or obese (30-35 kg/m2) patients undergoing elective liposuction were utilized. Cells were differentiated into adipocytes, co-cultured with RPMI 8226 or U266B1 multiple myeloma cell lines, and treated with standard MM therapies, including bortezomib or a triple combination of bortezomib, dexamethasone, and lenalidomide. We found that adipocytes from overweight and obese individuals increased cell adhesion-mediated drug resistance (CAM-DR) survival signals in MM cells, and P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) drug transporter expression. Further, co-culture enhanced in vitro angiogenesis, MMP-2 activity, and protected MM cells from drug-induced decreases in viability. In summary, we provide an underlying mechanism by which obesity can impair the drug response to MM and allow for recurrence and/or disease progression.

Targeting integrins in drug-resistant acute myeloid leukaemia.

Acute myeloid leukaemia (AML) continues to have a poor prognosis, warranting new therapeutic strategies. The bone marrow (BM) microenvironment consists of niches that interact with not only normal haematopoietic stem cells (HSC) but also leukaemia cells like AML. There are many adhesion molecules in the BM microenvironment; therein, integrins have been of central interest. AML cells express integrins that bind to ligands in the microenvironment, enabling adhesion of leukaemia cells in the microenvironment, thereby initiating intracellular signalling pathways that are associated with cell migration, cell proliferation, survival, and drug resistance that has been described to mediate cell adhesion-mediated drug resistance (CAM-DR). Identifying and targeting integrins in AML to interrupt interactions with the microenvironment have been pursued as a strategy to overcome CAM-DR. Here, we focus on the BM microenvironment and review the role of integrins in CAM-DR of AML and discuss integrin-targeting strategies. LINKED ARTICLES: This article is part of a themed issue on Cancer Microenvironment and Pharmacological Interventions. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.2/issuetoc.

Abstract B001: Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo

Abstract Bladder, colon, gastric, prostate, and uterine cancers originate from the organ’s epithelium, and each organ is surrounded by smooth muscle. In prostate cancer, organ-confined tumors, without extracapsular extension (ECE) through muscle, have a 5-year survival rate of 99% compared to 31% for metastatic disease. Previously, we modeled tumor cluster ECE and reported that a CRISPR-Cas9 integrin mutation (α6AA) blocked ECE by switching from a cell-ECM to a cell-cell biophysical phenotype. Since tumors are mosaics and migrate as heterogeneous drug-resistant populations, we tested the functional heterogeneity of the two biophysical cell phenotypes in ECE. Experimental procedures included the in vivo mouse xenograft model of ECE as a functional endpoint of muscle invasion, the MTT assay for cell survival, and using electric cell impedance sensing (ECIS) measurements to directly compare the biophysical properties of tumor cells expressing the invasion permissive α6WT integrin, cells expressing only the α6AA mutation, or a mixed population expressing both integrin types. The α6AA integrin mutation or α6KO knockout produced cells unable to invade into and through the smooth muscle of the mouse as compared to the α6WT integrin cells, as previously reported by us. The new unpublished results are that cell adhesion mediated drug resistance was detected in the α6AA population since their LD50 to Bortezomib, Gemcitabine, and Taxotere was shifted to 33.71nM, 64.66nM, and 5.77nM respectively from 16.29nM, 31.74nM, and 3.52nM in the α6WT cells as measured by MTT assay at 72 hours of incubation. In contrast, the α6AA cells were sensitized to the NAMPT inhibitor, FK866, with LD50 shifting from 27.46nM in α6WT to 7.354nM in the α6AA cells. A mixture of α6AA and α6WT cells allowed invasion into and through the muscle in the mouse of the mutant α6AA integrin cells that could not invade on their own. Examining the live cellular biophysical parameters revealed that the integrin mutation produced a 4-fold increase in cell-cell resistance (400Hz measurement) and a 12-fold recovery time delay in re-establishing a cell-cell resistance monolayer after wounding, with response complexity decreased two-fold. Cell-ECM resistance (40,000Hz measurement) in α6AA versus α6WT population was 1.5-fold decreased with a 6-fold recovery time delay in the cell-ECM resistance monolayer and response complexity decreased 2-fold. A heterogenous population containing up to a 4:1 proportion of α6AA to α6WT cells synergistically delayed cell-cell recovery time of the wounded monolayer up to 6-fold compared to the α6WT cells. Conversely, recovery of the wounded cell-ECM resistance monolayer with 1:1 or 4:1 mixture of α6AA cell only delayed recovery time by 1.5–1.7-fold. Taken together, these data suggest that a heterogenous tumor population provides functional advantages for drug-resistant tumor cells to invade and traverse the muscle barrier. Current work is ongoing to optimize the drug combinations to eliminate both phenotypes in the invasive tumor network and prevent ECE. Citation Format: Kendra D. Marr, Jaime M.C. Gard, William L. Harryman, Elijah J. Keeswood, Allan I. Paxson, Charles Wolgemuth, Lori A. Hazlehurst, Raymond B. Nagle, Anne E. Cress. Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B001.

Integrins and the Metastasis-like Dissemination of Acute Lymphoblastic Leukemia to the Central Nervous System.

Acute lymphoblastic leukemia (ALL) disseminates with high prevalence to the central nervous system (CNS) in a process resembling aspects of the CNS surveillance of normal immune cells as well as aspects of brain metastasis from solid cancers. Importantly, inside the CNS, the ALL blasts are typically confined within the cerebrospinal fluid (CSF)-filled cavities of the subarachnoid space, which they use as a sanctuary protected from both chemotherapy and immune cells. At present, high cumulative doses of intrathecal chemotherapy are administered to patients, but this is associated with neurotoxicity and CNS relapse still occurs. Thus, it is imperative to identify markers and novel therapy targets specific to CNS ALL. Integrins represent a family of adhesion molecules involved in cell-cell and cell-matrix interactions, implicated in the adhesion and migration of metastatic cancer cells, normal immune cells, and leukemic blasts. The ability of integrins to also facilitate cell-adhesion mediated drug resistance, combined with recent discoveries of integrin-dependent routes of leukemic cells into the CNS, have sparked a renewed interest in integrins as markers and therapeutic targets in CNS leukemia. Here, we review the roles of integrins in CNS surveillance by normal lymphocytes, dissemination to the CNS by ALL cells, and brain metastasis from solid cancers. Furthermore, we discuss whether ALL dissemination to the CNS abides by known hallmarks of metastasis, and the potential roles of integrins in this context.

The Role of Integrin Beta-1 in the Chemoresistance of Acute Myeloid Leukemia

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive disease that has a poor prognosis with a survival rate of 30-40%. The bone marrow microenvironment provides protection to AML cells from chemotherapy and thus confers cell adhesion-mediated drug resistance (CAM-DR) and promotes relapse. Integrins are key anchor molecules in CAM-DR of leukemia cells; they are heterodimeric adhesion molecules on the cell surface consisting of an alpha and a beta unit. Upregulated integrin alpha 4 and 6 expression is associated with poor survival in leukemias. We hypothesize that the targeting of integrin beta 1 (ITGB1, β1, or CD29) will also affect the integrin alpha units as they are heterodimer partners. This could be a superior approach to overcome CAM-DR in AML than just targeting the individual alpha units. METHODS: ITGB1 gene was knocked out via CRISPR-Cas9 using 2 guide RNAs in AML cell lines and expression of integrin subunits was profiled by flow cytometry. AML cell lines were treated in vitro with the anti-human ITGB1 monoclonal antibody OS2966 in combination with Ara-C chemotherapy and we determined effects on cell viability by flow cytometry and on cell signaling using RNA-sequencing. Using human patient-derived AML xenograft mouse models, we tested if blockade of ITGB1 combined with chemotherapy can overcome CAM-DR in vivo. Mice were treated with control total Ig (10 mg/kg), OS2966 (10 mg/kg), total Ig + Ara-C (600 mg/kg), or OS2966 + Ara-C twice a week for two weeks. RESULTS: Analysis of patient-derived AML cells at diagnosis and relapse, and of AML cell lines, showed high expression of ITGB1 and varying levels of other alpha and beta units. Loss-of-function studies of ITGB1 using CRISPR/Cas9-mediated knockout of ITGB1 in MLL-rearranged and FLT3-ITD+ AML cell lines MV4-11 and MOLM-13, and in AML cell line U937, revealed that surface expression of integrin alphas 3, 4, 5 and 6 were markedly decreased as determined by flow cytometry analysis. ITGB2 expression was high in all 3 AML cell lines and not affected by ITGB1 deletion. Remarkably, ITGB3 was downregulated upon ITGB1 deletion in 2 out of 3 AML cell lines, whereas the 3rd AML cell line did not express any ITGB3. As a result of the ITGB1 deletion, AML cells showed less adhesion to murine bone marrow stroma (OP9) cells and to extracellular matrix proteins. We found that inhibition of surface ITGB1 on human AML using OS2966 de-adhered the AML cells from OP9 cells. Moreover, ITGB1 blockade using OS2966 synergized with Ara-C in vitro and significantly decreased cell viability compared to single agent controls in human AML cell lines U937 and MV4-11. RNA sequencing data suggest that the NFkB pathway is significantly dysregulated in the combination treatment Ara-C and OS2966 compared to Ara-C alone. To test ITGB1 inhibition in vivo, a patient-derived AML sample, HEM279 (Fig. 1A), and AML cell line, U937 (Fig. 1B), were engrafted into NSG-SGM3 mice. Leukemic mice were treated with single agents including control total Ig, OS2966, or Ara-C; and with total Ig in combination with Ara-C or OS2966. In both xenograft models, the combination treatment of OS2966 with Ara-C significantly prolonged survival of leukemic mice compared to Ara-C only: HEM279: MST 115 +/- 15.4 days vs 73.1 +/- 5.2 days post-AML injection; p=0.0005; U937: MST 170 days vs 32 days post-AML injection; p=0.0004; 50% of the mice engrafted with U937 and treated with OS2966 with Ara-C survived until day 290 post-AML injection, which is when the experiment was terminated. CONCLUSIONS: Integrin alpha subunit expression is dependent on ITGB1 expression, supporting our hypothesis that targeting integrin beta 1 affects alpha integrin units. ITGB1 inhibition with OS2966 combined with chemotherapy significantly decreases cell viability of AML cell lines in vitro and prolongs survival of xenograft mice in vivo. Our findings will allow for better understanding of the role of ITGB1 in drug resistance of AML and of ITGB1 as a preclinical target in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting CAM-DR and Mitochondrial Transfer for the Treatment of Multiple Myeloma.

The prognosis of patients with multiple myeloma (MM) has improved dramatically with the introduction of new therapeutic drugs, but the disease eventually becomes drug-resistant, following an intractable and incurable course. A myeloma niche (MM niche) develops in the bone marrow microenvironment and plays an important role in the drug resistance mechanism of MM. In particular, adhesion between MM cells and bone marrow stromal cells mediated by adhesion molecules induces cell adhesion-mediated drug resistance (CAM-DR). Analyses of the role of mitochondria in cancer cells, including MM cells, has revealed that the mechanism leading to drug resistance involves exchange of mitochondria between cells (mitochondrial transfer) via tunneling nanotubes (TNTs) within the MM niche. Here, we describe the discovery of these drug resistance mechanisms and the identification of promising therapeutic agents primarily targeting CAM-DR, mitochondrial transfer, and TNTs.

Transformable ECM Deprivation System Effectively Suppresses Renal Cell Carcinoma by Reversing Anoikis Resistance and Increasing Chemotherapy Sensitivity (Adv. Mater. 43/2022)

Renal Cell Carcinoma In article number 2203518, Ziqi Wang, Lei Wang, Hao Wang, Wanhai Xu, and co-workers report a transformable extracellular matrix (ECM) deprivation system (EDS) that targets fibronectin for depriving the ECM of renal cell carcinoma (RCC). The cage-like nanofibers formed by the EDS deprive the ECM of RCC cells, which efficaciously reverses anoikis resistance and ameliorates the RCC's cell-adhesion-mediated drug resistance (CAM-DR).

Transformable ECM Deprivation System Effectively Suppresses Renal Cell Carcinoma by Reversing Anoikis Resistance and Increasing Chemotherapy Sensitivity.

Extracellular matrix (ECM) is crucial in various biological functions during tumor progression, including induction of anoikis resistance and cell adhesion-mediated drug resistance (CAM-DR). Fibronectin (FN) is a vital ECM component with direct regulatory effects on ECM-mediated anoikis resistance and CAM-DR, making it an attractive and innovative therapeutic target for depriving ECM in tumor tissue. Herein, an ECM deprivation system (EDS) is developed based on FN targeting self-assembly peptide for constructing nanofibers in the ECM of renal cell carcinoma (RCC), which contributes to: i) targeting and recognizing FN to form nanofibers for long-term retention in ECM, ii) reversing anoikis resistance via arresting the FN signaling pathway, and iii) serving as a drug-loading platform for sensitizing chemotherapy by ameliorating CAM-DR. The results reveal that EDS significantly reverses anoikis resistance of RCC cells by inhibiting the phosphorylation of FAK, a positive regulator of the FN signaling pathway. Meanwhile, EDS serves as a chemotherapy-sensitizer of cancer, exerting significant synergistic effects with doxorubicin (DOX). In vivo validation experiments show that EDS effectively suppresses metastasis and tumor growth with chemotherapy resistance. Collectively, the innovative EDS notably inhibits the tumor-promoting effect of ECM and may provide a novel approach for suppressing ECM and enhancing chemo-drug sensitivity.

Inhibition of lysyl oxidase-like 2 overcomes adhesion-dependent drug resistance in the collagen-enriched liver cancer microenvironment.

The tumor microenvironment (TME) is considered to be one of the vital mediators of tumor progression. Extracellular matrix (ECM), infiltrating immune cells, and stromal cells collectively constitute the complex ecosystem with varied biochemical and biophysical properties. The development of liver cancer is strongly tied with fibrosis and cirrhosis that alters the microenvironmental landscape, especially ECM composition. Enhanced deposition and cross‐linking of type I collagen are frequently detected in patients with liver cancer and have been shown to facilitate tumor growth and metastasis by epithelial‐to‐mesenchymal transition. However, information on the effect of collagen enrichment on drug resistance is lacking. Thus, the present study has comprehensively illustrated phenotypical and mechanistic changes in an in vitro mimicry of collagen‐enriched TME and revealed that collagen enrichment could induce 5‐fluorouracil (5FU) and sorafenib resistance in liver cancer cells through hypoxia‐induced up‐regulation of lysyl oxidase‐like 2 (LOXL2). LOXL2, an enzyme that facilitates collagen cross‐linking, enhances cell adhesion‐mediated drug resistance by activating the integrin alpha 5 (ITGA5)/focal adhesion kinase (FAK)/phosphoinositide 3‐kinase (PI3K)/rho‐associated kinase 1 (ROCK1) signaling axis. Conclusion: We demonstrated that inhibition of LOXL2 in a collagen‐enriched microenvironment synergistically promotes the efficacy of sorafenib and 5FU through deterioration of focal adhesion signaling. These findings have clinical implications for developing LOXL2‐targeted strategies in patients with chemoresistant liver cancer and especially for those patients with advanced fibrosis and cirrhosis.

Integrin Signaling Shaping BTK-Inhibitor Resistance.

Integrins are adhesion molecules that function as anchors in retaining tumor cells in supportive tissues and facilitating metastasis. Beta1 integrins are known to contribute to cell adhesion-mediated drug resistance in cancer. Very late antigen-4 (VLA-4), a CD49d/CD29 heterodimer, is a beta1 integrin implicated in therapy resistance in both solid tumors and haematological malignancies such as chronic lymphocytic leukemia (CLL). A complex inside-out signaling mechanism activates VLA-4, which might include several therapeutic targets for CLL. Treatment regimens for this disease have recently shifted towards novel agents targeting BCR signaling. Bruton’s tyrosine kinase (BTK) is a component of B cell receptor signaling and BTK inhibitors such as ibrutinib are highly successful; however, their limitations include indefinite drug administration, the development of therapy resistance, and toxicities. VLA-4 might be activated independently of BTK, resulting in an ongoing interaction of CD49d-expressing leukemic cells with their surrounding tissue, which may reduce the success of therapy with BTK inhibitors and increases the need for alternative therapies. In this context, we discuss the inside-out signaling cascade culminating in VLA-4 activation, consider the advantages and disadvantages of BTK inhibitors in CLL and elucidate the mechanisms behind cell adhesion-mediated drug resistance.

Mechanisms of Cell Adhesion Molecules in Endocrine-Related Cancers: A Concise Outlook.

Chemotherapy is a critical treatment for endocrine-related cancers; however, chemoresistance and disease recurrence remain a challenge. The interplay between cancer cells and the tumor microenvironment via cell adhesion molecules (CAMs) promotes drug resistance, known as cell adhesion-mediated drug resistance (CAM-DR). CAMs are cell surface molecules that facilitate cell-to-cell or cell-to-extracellular matrix binding. CAMs exert an adhesion effect and trigger intracellular signaling that regulates cancer cell stemness maintenance, survival, proliferation, metastasis, epithelial–mesenchymal transition, and drug resistance. To understand these mechanisms, this review focuses on the role of CD44, cadherins, selectins, and integrins in CAM-DR in endocrine-related cancers.

Significance of a tumor microenvironment-mediated P65-miR-30a-5p-BCL2L11 amplification loop in multiple myeloma

Despite significant progress in the treatment of myeloma, multiple myeloma (MM) remains an incurable hematological malignancy due to cell adhesion-mediated drug resistance (CAM-DR) phenotype. However, data on the molecular mechanisms underlying the CAM-DR remains scanty. Here, we identified a miRNA-mRNA regulatory network in myeloma cells that are directly adherent to bone marrow stromal cells (BMSCs). Our data showed that the BMSCs up-regulated miR-30a-5p and down-regulated BCL2L11 at both mRNA and protein level in the myeloma cells. Besides, luciferase reporter genes demonstrated direct interaction between miR-30a-5p and BCL2L11 gene. Moreover, the BMSCs activated NF-ΚB signaling pathway in myeloma cells and the NF-κB P65 was shown to directly bind the miR-30a-5p promoter region. Moreover, suppression of the miR-30a-5p or upregulation of the BCL2L11 promoted apoptosis of the myeloma cells independent of the BMSCs, thus suggesting clinical significance of miR-30a-5p inhibitor and PLBCL2L11 plasmid in CAM-DR. Together, our data demonstrated the role of P65-miR-30a-5p-BCL2L11 loop in CAM-DR myeloma cells. These findings give new insights into the role of tumor microenvironment in the treatment of patients with myeloma.

PI3K Targeting in Non-solid Cancer.

Despite the therapeutic progress, relapse remains a major problem in the treatment of acute lymphoblastic leukemia (ALL). Most leukemia cells that survive chemotherapy are found in the bone marrow (BM), thus resistance to chemotherapy and other treatments may be partially attributed to pro-survival signaling to leukemic cells mediated by leukemia cell-microenvironment interactions. Adhesion of leukemia cells to BM stromal cells may lead to cell adhesion-mediated drug resistance (CAM-DR) mediating intracellular signaling changes that support survival of leukemia cells. In ALL and chronic lymphocytic leukemia (CLL), adhesion-mediated activation of the PI3K/AKT signaling pathway has been shown to be critical in CAM-DR. PI3K targeting inhibitors have been approved for CLL and have been evaluated preclinically in ALL. However, PI3K inhibition has yet to be approved for clinical use in ALL. Here, we review the role of PI3K signaling for normal hematopoietic and leukemia cells and summarize preclinical inhibitors of PI3K in ALL.

In vitro and in vivo effects of AVA4746, a novel competitive antagonist of the ligand binding of VLA-4, in B-cell acute lymphoblastic leukemia.

Treatment of resistant or recurrent acute lymphoblastic leukemia (ALL) remains a challenge. It was previously demonstrated that the adhesion molecule integrin α4, referred to hereafter as α4, mediates the cell adhesion-mediated drug resistance (CAM-DR) of B-cell ALL by binding to vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stroma. In addition, it was previously observed that the blockade of α4 with natalizumab or inhibition using the small molecule antagonist TBC3486 sensitized relapsed ALL cells to chemotherapy. However, α4-targeted therapy is not clinically available for the treatment of leukemia to date. In the present study, the use of a novel non-peptidic small molecule integrin α4 antagonist, AVA4746, as a potential new approach to combat drug-resistant B-ALL was explored. An in vitro co-culture = model of primary B-ALL cells and an in vivo xenograft model of patient-derived B-ALL cells were utilized for evaluation of AVA4746. VLA-4 conformation activation, cell adhesion/de-adhesion, endothelial tube formation, in vivo leukemia cell mobilization and survival assays were performed. AVA4746 exhibited high affinity for binding to B-ALL cells, where it also efficiently blocked ligand-binding to VCAM-1. In addition, AVA4746 caused the functional de-adhesion of primary B-ALL cells from VCAM-1. Inhibition of α4 using AVA4746 also prevented angiogenesis in vitro and when applied in combination with chemotherapy consisting of Vincristine, Dexamethasone and L-asparaginase, it prolonged the survival of ~33% of the mice in an in vivo xenograft model of B-ALL. These data implicate the potential of targeting the α4-VCAM-1 interaction using AVA4746 for the treatment of drug-resistant B-lineage ALL.

Second Treatment Free Remission after Combination Therapy with Ruxolitinib Plus Tyrosine Kinase Inhibitors in Chronic Phase Chronic Myeloid Leukemia (CML)

Second Treatment Free Remission after Combination Therapy with Ruxolitinib Plus Tyrosine Kinase Inhibitors in Chronic Phase Chronic Myeloid Leukemia (CML)

N-Cadherin Stabilizes β-Catenin and Promotes β-Catenin/TCF Transcriptional Activation and Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma

Introduction:Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM.Materials and Methods:We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described.Results:We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM.Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. DisclosuresDavies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.