Rajasekharan Somasundaram

Acquired Resistance to BRAF Inhibitors Mediated by a RAF Kinase Switch in Melanoma Can Be Overcome by Cotargeting MEK and IGF-1R/PI3K

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)⁶⁰⁰(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.

Immunomodulatory activity of monoclonal anti-idiotypic antibody to anti-colorectal carcinoma antibody CO17-1A in animals and patients.

Monoclonal anti-idiotypic antibody (Ab2) VF2 was derived from rats immunized with anti-colorectal carcinoma (anti-CRC) monoclonal antibody (Ab1) CO17-1A. In rabbits the Ab2 induced anti anti-idiotypic antibodies (Ab3) that shared idiotopes with the Ab1, bound to the same epitope on CRC cells as Ab1, and bound to the isolated CO17-1A antigen. Monoclonal Ab2 VF2 was superior to the previously described polyclonal goat Ab2 against Ab1 CO17-1A in its capacity to elicit humoral immunity in animals. Ab2 VF2 also induced a specific delayed-type hypersensitivity (DTH) response to challenge with irradiated CO17-1A antigen-positive human CRC cells in mice. Of nine CRC patients immunized with aluminum hydroxide-precipitated Ab2 VF2, six developed antibodies that bound to Ab2, but only three patients developed Ab3 that bound to idiotypic determinants on Ab2. However, the Ab3 did not bind to CO17-1A antigen-positive CRC cells. In contrast, in a previously described trial with polyclonal goat Ab2 to Ab1 CO17-1A, most of the patients developed anti-CRC antibodies. Four of the nine patients immunized with Ab2 VF2 developed DTH responses to intradermal challenge with the Ab2, and in one patient DTH was both Ab2- and antigen-specific. Peripheral blood mononu-clear cells of the four DTH-reactive patients did not proliferate in response to in vitro stimulation with either Ab2 or antigen. These studies demonstrate that the immunomodulatory activity of monoclonal Ab2 VF2 in animals is only in part predictive of its activity in patients.

Migration of cytotoxic T lymphocytes toward melanoma cells in three‐dimensional organotypic culture is dependent on CCL2 and CCR4

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor‐positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patients CTL toward autologous tumor cells has been studied in a novel three‐dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti‐CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2‐transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.

A stress-induced early innate response causes multidrug tolerance in melanoma

Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics leading to permanent resistance are poorly understood but are important to design better treatments. Here we show that drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multidrug resistance, termed induced drug-tolerant cells (IDTCs). Transition into the IDTC state seems to be an inherent stress reaction for survival toward unfavorable environmental conditions or drug exposure. The response comprises chromatin remodeling, activation of signaling cascades and markers implicated in cancer stemness with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced aldehyde dehydrogenase (ALDH) activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug-resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

CXC Chemokine Ligand 12 (Stromal Cell-Derived Factor 1α) and CXCR4-Dependent Migration of CTLs toward Melanoma Cells in Organotypic Culture

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient’s CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1α) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

Abstract In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freunds complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freunds adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector.

Melanoma-derived conditioned media efficiently induce the differentiation of monocytes to macrophages that display a highly invasive gene signature.

The presence of tumor‐associated macrophages (TAMs) in melanomas is correlated with a poor clinical prognosis. However, there is limited information on the characteristics and biological activities of human TAMs in melanomas. In this study, we developed an in vitro method to differentiate human monocytes to macrophages using modified melanoma‐conditioned medium (MCM). We demonstrate that factors from MCM‐induced macrophages (MCMI‐Mφ) express both M1‐Mφ and M2‐Mφ markers and inhibit melanoma‐specific T‐cell proliferation. Furthermore, microarray analyses reveal that the majority of genes up‐regulated in MCMI‐Mφ are associated with tumor invasion. The most strikingly up‐regulated genes are CCL2 and MMP‐9. Consistent with this, blockade of both CCL‐2 and MMPs diminish MCMI‐Mφ‐induced melanoma invasion. Finally, we demonstrated that both MCMI‐Mφ and in vivo TAMs express the pro‐invasive, melanoma‐associated gene, glycoprotein non‐metastatic melanoma protein B. Our study provides a framework for understanding the mechanisms of cross‐talk between TAMs and melanoma cells within the tumor microenvironment.

Intratumoral Heterogeneity as a Therapy Resistance Mechanism: Role of Melanoma Subpopulations

Malignant melanoma is an aggressive form of skin cancer whose incidence continues to increase worldwide. Increased exposure to sun, ultraviolet radiation, and the use of tanning beds can increase the risk of melanoma. Early detection of melanomas is the key to successful treatment mainly through surgical excision of the primary tumor lesion. But in advanced stage melanomas, once the disease has spread beyond the primary site to distant organs, the tumors are difficult to treat and quickly develop resistance to most available forms of therapy. The advent of molecular and cellular techniques has led to a better characterization of tumor cells revealing the presence of heterogeneous melanoma subpopulations. The discovery of gene mutations and alterations of cell-signaling pathways in melanomas has led to the development of new targeted drugs that show dramatic response rates in patients. Single-agent therapies generally target one subpopulation of tumor cells while leaving others unharmed. The surviving subpopulations will have the ability to repopulate the original tumors that can continue to progress. Thus, a rational approach to target multiple subpopulations of tumor cells with a combination of drugs instead of single-agent therapy will be necessary for long-lasting inhibition of melanoma lesions. In this context, the recent development of immune checkpoint reagents provides an additional armor that can be used in combination with targeted drugs to expand the presence of melanoma reactive T cells in circulation to prevent tumor recurrence.

Chemokines and the microenvironment in neuroectodermal tumor-host interaction

Chemokines and chemokine receptors play an important role in immune homeostasis and surveillance. Altered or defective expression of chemokines and/or chemokine receptors could lead to a disease state including autoimmune disorder or cancer. Tumors from glioblastoma, melanoma, and neuroblastoma secrete high levels of chemokines that can promote tumor growth and progression or induce stromal cells present in the tumor microenvironment to produce cytokines or chemokines which, in turn, can regulate angiogenesis, tumor growth, and metastasis. On the other hand, chemokines secreted by tumor or stromal cells can also attract leukocytes such as dendritic cells, macrophages, neutrophils, and lymphocytes which may downmodulate tumor growth. New therapies that are aimed at limiting tumor growth and progression by attracting immune effector cells to the tumor site with chemokines may hold the key to the successful treatment of cancer, although this approach may be hampered by possible tumor growth-stimulating effects of chemokines.

Targeting CD20 in Melanoma Patients at High Risk of Disease Recurrence

Melanomas contain distinct cell subpopulations. Several of these subpopulations, including one expressing CD20, may harbor stem cell-like or tumor-initiating characteristics. We hypothesized that patients at high risk of disease recurrence could benefit from an adjuvant anti-CD20 therapy. Therefore, we initiated a small pilot trial to study the effect of the anti-CD20 antibody rituximab in a group of melanoma patients with stage IV metastatic disease who had been rendered without evident disease by way of surgery, chemotherapy and/or radiation therapy. The major objective was safety, while secondary objectives were description of recurrence-free intervals (RFI) and overall survival (OS). Nine patients received rituximab at 375 mg/m(2) qw for 4 weeks followed by a maintenance therapy every 8 weeks. Treatment was discontinued after 2 years or with disease recurrence. Treatment was well tolerated. After a median observation of 42 months, the median neither of RFI nor of OS has been reached. Despite therapy that ended after 2 years, six out of nine patients are still alive and five of them are recurrence-free. Though the patient number is too small for definitive conclusions, our data may represent a first example of the potential therapeutic value of targeting CD20(+) cell populations-at least for a subset of patients.