Kenneth H. Shain

Regulation of the innate and adaptive immune responses by Stat-3 signaling in tumor cells.

Although tumor progression involves processes such as tissue invasion that can activate inflammatory responses, the immune system largely ignores or tolerates disseminated cancers. The mechanisms that block initiation of immune responses during cancer development are poorly understood. We report here that constitutive activation of Stat-3, a common oncogenic signaling pathway, suppresses tumor expression of proinflammatory mediators. Blocking Stat-3 in tumor cells increases expression of proinflammatory cytokines and chemokines that activate innate immunity and dendritic cells, leading to tumor-specific T-cell responses. In addition, constitutive Stat-3 activity induces production of pleiotropic factors that inhibit dendritic cell functional maturation. Tumor-derived factors inhibit dendritic cell maturation through Stat-3 activation in progenitor cells. Thus, inhibition of antitumor immunity involves a cascade of Stat-3 activation propagating from tumor to dendritic cells. We propose that tumor Stat-3 activity can mediate immune evasion by blocking both the production and sensing of inflammatory signals by multiple components of the immune system.

β1 Integrin Adhesion Enhances IL-6–Mediated STAT3 Signaling in Myeloma Cells: Implications for Microenvironment Influence on Tumor Survival and Proliferation

The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of multiple myeloma (MM) cells; however, in vivo, these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via beta1 integrins, stimulated with IL-6, or treated with the two combined. Although G(1)-S cell cycle arrest associated with FN adhesion was overcome when IL-6 was added, the cell adhesion-mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in signal transducers and activators of transcription 3 (STAT3) phosphorylation, nuclear translocation, and DNA binding, compared with either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN before stimulation with IL-6, relative to nonadherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by beta1 integrin and gp130 confers an increased survival advantage to MM cells.

Stat3 Activity in Melanoma Cells Affects Migration of Immune Effector Cells and Nitric Oxide-Mediated Antitumor Effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-α and IFN-β, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-α in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.

Adhesion-Mediated Intracellular Redistribution of c-Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Like Inhibitory Protein-Long Confers Resistance to CD95-Induced Apoptosis in Hematopoietic Cancer Cell Lines

Evasion of immune surveillance is a key step in malignant progression. Interactions between transformed hematopoietic cells and their environment may initiate events that confer resistance to apoptosis and facilitate immune evasion. In this report, we demonstrate that β1 integrin-mediated adhesion to fibronectin inhibits CD95-induced caspase-8 activation and apoptosis in hematologic tumor cell lines. This adhesion-dependent inhibition of CD95-mediated apoptosis correlated with enhanced c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long (c-FLIPL) cytosolic solubility compared with nonadhered cells. Cytosolic c-FLIPL protein preferentially associated with cytosolic Fas-associated death domain protein (FADD) and localized to the death-inducing signal complex after CD95 ligation in adherent cells. The incorporation of c-FLIPL in the death-inducing signal complex prevented procaspase-8 processing and activation of the effector phase of apoptosis. Adhesion to fibronectin increased c-FLIPL cytosolic solubility and availability for FADD binding by redistributing c-FLIPL from a preexisting membrane-associated fraction. Increased cytosolic availability of c-FLIPL for FADD binding was not related to increased levels of RNA or protein synthesis. These data show that adhesion of anchorage-independent cells to fibronectin provides a novel mechanism of resistance to CD95-mediated programmed cell death by regulating the cellular localization and availability of c-FLIPL.

The tumor microenvironment as a determinant of cancer cell survival: A possible mechanism for de novo drug resistance

The influence of the microenvironment in the pathogenesis and progression of human cancer has traditionally been considered in the context of solid tumors. More recently, evidence has been accumulating to support the role of the bone marrow microenvironment in hematologic malignancies as well, particularly in multiple myeloma. This review focuses on myeloma as a model to demonstrate that the bone marrow microenvironment provides a sanctuary against programmed cell death and promotes tumor cell survival and progression. Additionally, the protective effects of the bone marrow milieu may confer a protection from cytotoxic drugs, allowing the emergence of drug-resistant tumors. These advances may assist in the design of novel therapeutic approaches to enhance the efficacy of standard chemotherapeutic drugs.

Targeting the Fanconi Anemia/BRCA Pathway Circumvents Drug Resistance in Multiple Myeloma

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-kappaB subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of IkappaB kinase alpha and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed multiple myeloma cells. Inhibiting NF-kappaB by small interfering RNA, blocking the IkappaB kinase complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-kappaB transcriptionally regulates the FA/BRCA pathway and provide evidence for targeting Fanconi anemia-mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients.

Randomized multicenter phase 2 study of pomalidomide, cyclophosphamide, and dexamethasone in relapsed refractory myeloma

Pomalidomide and low-dose dexamethasone (PomDex) is standard treatment of lenalidomide refractory myeloma patients who have received >2 prior therapies. We aimed to assess the safety and efficacy of the addition of oral weekly cyclophosphamide to standard PomDex. We first performed a dose escalation phase 1 study to determine the recommended phase 2 dose of cyclophosphamide in combination with PomDex (arm A). A randomized, multicenter phase 2 study followed, enrolling patients with lenalidomide refractory myeloma. Patients were randomized (1:1) to receive pomalidomide 4 mg on days 1 to 21 of a 28-day cycle in combination with weekly dexamethasone (arm B) or pomalidomide, dexamethasone, and cyclophosphamide (PomCyDex) 400 mg orally on days 1, 8, and 15 (arm C). The primary end point was overall response rate (ORR). Eighty patients were enrolled (10 in phase 1 and 70 randomized in phase 2: 36 to arm B and 34 to arm C). The ORR was 38.9% (95% confidence interval [CI], 23-54.8%) and 64.7% (95% CI, 48.6-80.8%) for arms B and C, respectively (P = .035). As of June 2015, 62 of the 70 randomized patients had progressed. The median progression-free survival (PFS) was 4.4 (95% CI, 2.3-5.7) and 9.5 months (95% CI, 4.6-14) for arms B and C, respectively (P = .106). Toxicity was predominantly hematologic in nature but was not statistically higher in arm C. The combination of PomCyDex results in a superior ORR and PFS compared with PomDex in patients with lenalidomide refractory multiple myeloma. The trial was registered at www.clinicaltrials.gov as #NCT01432600.

Bone Marrow Stroma Confers Resistance to Apo2 Ligand/TRAIL in Multiple Myeloma in Part by Regulating c-FLIP

Apo2 ligand (Apo2L)/TRAIL induces apoptosis of cancer cells that express the specific receptors while sparing normal cells. Because the tumor microenvironment protects myeloma from chemotherapy, we investigated whether hemopoietic stroma induces resistance to Apo2L/TRAIL apoptosis in this disease. Apo2L/TRAIL-induced death was diminished in myeloma cell lines (RPMI 8226, U266, and MM1s) directly adhered to a human immortalized HS5 stroma cell line but not adhered to fibronectin. In a Transwell assay, with myeloma in the upper well and HS5 cells in the lower well, Apo2L/TRAIL apoptosis was reduced when compared with cells exposed to medium in the lower well. Using HS5 and myeloma patients’ stroma-conditioned medium, we determined that soluble factor(s) produced by stroma–myeloma interactions are responsible for a reversible Apo2/TRAIL apoptosis resistance. Soluble factor(s) attenuated procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase cleavage and diminished mitochondrial membrane potential changes without affecting Bcl-2 family proteins and/or Apo2L/TRAIL receptors. Soluble factor(s) increased the baseline levels of the anti-apoptotic protein c-FLIP in all cell lines tested. Inhibition of c-FLIP by means of RNA interference increased Apo2/TRAIL sensitivity in RPMI 8226 cells. Unlike direct adhesion to fibronectin, soluble factor(s) have no impact on c-FLIP redistribution within cellular compartments. Cyclohexamide restored Apo2L/TRAIL sensitivity in association with down-regulation of c-FLIP, suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factor(s) to regulate c-FLIP. Additionally, IL-6 conferred resistance to Apo2L/TRAIL-mediated apoptosis in association with increased c-FLIP levels. In conclusion, the immune cytotoxic effect of Apo2L/TRAIL can be restored at least in part by c-FLIP pathway inhibitors.

Stemness of B-cell Progenitors in Multiple Myeloma Bone Marrow

Purpose: In myeloma, B cells and plasma cells show a clonal relationship. Clonotypic B cells may represent a tumor-initiating compartment or cancer stem cell responsible for minimal residual disease in myeloma. Experimental Design: We report a study of 58 patients with myeloma at time of diagnosis or relapse. B cells in bone marrow were evaluated by multicolor flow cytometry and sorting. Clonality was determined by light chain and/or immunoglobulin chain gene rearrangement PCR. We also determined aldehyde dehydrogenase activity and colony formation growth. Drug sensitivity was tested with conventional and novel agents. Results: Marrow CD19+ cells express a light chain identical to plasma cells and are therefore termed light chain restricted (LCR). The LCR B-cell mass is small in both newly diagnosed and relapsed patients (≤1%). Few marrow LCR B cells (∼10%) are CD19+/CD34+, with the rest being more differentiated CD19+/CD34− B cells. Marrow LCR CD19+ B cells exhibit enhanced aldehyde dehydrogenase activity versus healthy controls. Both CD19+/CD34+ and CD19+/CD34− cells showed colony formation activity, with colony growth efficiency optimized when stroma-conditioned medium was used. B-cell progenitors showed resistance to melphalan, lenalidomide, and bortezomib. Panobinostat, a histone deacetylase inhibitor, induced apoptosis of LCR B cells and CD138+ cells. LCR B cells are CD117, survivin, and Notch positive. Conclusions: We propose that antigen-independent B-cell differentiation stages are involved in disease origination and progression in myeloma. Furthermore, investigations of myeloma putative stem cell progenitors may lead to novel treatments to eradicate the potential reservoir of minimal residual disease. Clin Cancer Res; 18(22); 6155–68. ©2012 AACR.

Clonal haemopoiesis and therapy-related myeloid malignancies in elderly patients: a proof-of-concept, case-control study

BACKGROUND Clonal haemopoiesis of indeterminate potential (CHIP) is an age-associated genetic event linked to increased risk of primary haematological malignancies and increased all-cause mortality, but the prevalence of CHIP in patients who develop therapy-related myeloid neoplasms is unknown. We did this study to investigate whether chemotherapy-treated patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. METHODS We did a nested, case-control, proof-of-concept study to compare the prevalence of CHIP between patients with cancer who later developed therapy-related myeloid neoplasms (cases) and patients who did not develop these neoplasms (controls). We identified cases from our internal biorepository of 123 357 patients who consented to participate in the Total Cancer Care biobanking protocol at Moffitt Cancer Center (Tampa, FL, USA) between Jan 1, 2006, and June 1, 2016. We included all individuals who were diagnosed with a primary malignancy, were treated with chemotherapy, subsequently developed a therapy-related myeloid neoplasm, and were 70 years or older at either diagnosis. For inclusion in this study, individuals must have had a peripheral blood or mononuclear cell sample collected before the diagnosis of therapy-related myeloid neoplasm. Controls were individuals who were diagnosed with a primary malignancy at age 70 years or older and were treated with chemotherapy but did not develop therapy-related myeloid neoplasms. Controls were matched to cases in at least a 4:1 ratio on the basis of sex, primary tumour type, age at diagnosis, smoking status, chemotherapy drug class, and duration of follow-up. We used sequential targeted and whole-exome sequencing and described clonal evolution in cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available. The primary endpoint of this study was the development of therapy-related myeloid neoplasm and the primary exposure was CHIP. FINDINGS We identified 13 cases and 56 case-matched controls. The prevalence of CHIP in all patients (23 [33%] of 69 patients) was higher than has previously been reported in elderly individuals without cancer (about 10%). Cases had a significantly higher prevalence of CHIP than did matched controls (eight [62%] of 13 cases vs 15 [27%] of 56 controls, p=0·024; odds ratio 5·75, 95% CI 1·52-25·09, p=0·013). The most commonly mutated genes in cases with CHIP were TET2 (three [38%] of eight patients) and TP53(three [38%] of eight patients), whereas controls most often had TET2 mutations (six [40%] of 15 patients). In most (four [67%] of six patients) cases for whom paired CHIP and therapy-related myeloid neoplasm samples were available, the mean allele frequency of CHIP mutations had expanded by the time of the therapy-related myeloid neoplasm diagnosis. However, a subset of paired samples (two [33%] of six patients) had CHIP mutations that decreased in allele frequency, giving way to expansion of a distinct mutant clone. INTERPRETATION Patients with cancer who have CHIP are at increased risk of developing therapy-related myeloid neoplasms. The distribution of CHIP-related gene mutations differs between individuals with therapy-related myeloid neoplasm and those without, suggesting that mutation-specific differences might exist in therapy-related myeloid neoplasm risk. FUNDING Moffitt Cancer Center.