Aryl Hydrocarbon Receptor (AHR) Antagonism As a Transformative, Dual-Mechanism Novel Therapy for Multiple Myeloma

BACKGROUND: The aryl hydrocarbon receptor (AHR) is a transcription factor that regulates xenobiotic-metabolizing enzymes in response to exogenous polycyclic aromatic hydrocarbons. However, we have described a critical role for AHR in normal lymphocyte development and identified the functional expression of AHR in multiple myeloma (MM). Interest is growing in AHR as a novel anti-cancer therapeutic target in solid tumors, but very little comparatively is known about AHR in hematologic malignancies. Epidemiologic data suggest that exposure to the AHR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzene and dioxin may be linked to increased rates of MGUS and MM in Vietnam-era veterans as well as firefighters at the World Trade Center attacks in September, 2001. Whereas we have shown that AHR antagonism facilitates normal, healthy natural killer (NK) cell lymphocyte development and acquisition of cytolytic potential, herein, we show that AHR antagonism impairs MM cell viability. In addition, we characterize the direct effects of AHR antagonism on MM cells as well as complementary mechanisms by which the NK cell versus MM effect is augmented, further strengthening ongoing interest in targeting AHR as a novel anti-MM therapy. METHODS: The MM cell lines MM.1S, MM.1R, OPM-2, and U266 were obtained from ATCC. Primary cells from healthy donors and patients with MM were procured under IRB-approved protocols. AHR expression was studied by Western blot, flow cytometry and real time PCR. AHR function was assessed by expression of the downstream target CYP1A1 by real time PCR. Clinical MM outcomes were determined with data obtained from a publically available database. Cell proliferation and viability were studied by light microscopy (trypan blue exclusion) and Sytox. Immunophenotyping was conducted by flow cytometry. Cytotoxicity was measured by 51Cr release assay. The AHR antagonists CH233191 and CB7993113 and the high affinity AHR agonist 5,11-dihydroindolol[3,2-b]carbazole-6-carboxaldehyde (FICZ) were utilized to study the in vitro effects of AHR modulation. RESULTS: AHR transcript and protein are expressed in human NK cells, plasma cells and B cells, as well as MM cell lines and primary MM samples. AHR agonism increases transcription of CYP1A1 in MM cells whereas AHR antagonism suppresses CYP1A1; these observations confirm that AHR is functionally active in MM. AHR is abundantly expressed from the CD38brightCD27(+) MM stem cell compartment to primary plasma cell leukemia samples. MM cases with high AHR expression are associated with inferior survival as compared to MM cases with low AHR expression (median OS = 50 months versus not reached at 70 months, p = 0.00002). AHR stimulation reduces expression of CD38, CD56, and CD138 and upregulates CD10, CD11a, CD13, CD19, CD20, CD27, CD40, CD45, and CD117 consistent with MM clonogenic progenitor cells previously described as "stem-like" and drug resistant. AHR antagonism suppresses MM cell line and primary MM cell viability, including cytogenetically high-risk, plasma cell leukemia primary samples. These effects are preserved in 7- and 14-day washout experiments. AHR antagonism increases expression of CD38 (2.1-fold) and SLAMF7 (CS1, 1.54-fold) among other antigens. AHR antagonism enhances NK cell cytolytic potential by promoting expression of CD94, TRAIL, perforin and granzyme B, whereas in MM cells, AHR antagonism upregulates surface expression of NK cell activating ligands: CD112, CD54, ULBP1, ULBP2, ULBP3, ULBP4, CD155, MICA/B, and DR4 and DR5. Pretreatment of MM cell targets with an AHR antagonist enhances susceptibility to NK-cell cytotoxicity (p < 0.005) and augments NK-cell mediated ADCC in combination with daratumumab (p = 0.001) and elotuzumab (p = 0.001) versus controls. CONCLUSIONS: These data confirm functional expression of AHR in MM and may provide insight to growing epidemiologic data indicting a link between environmental exposure to aromatic hydrocarbons and subsequent risk of MM. In addition, as opposed to the direct, deleterious effects of AHR antagonism on MM cell viability, AHR antagonism promotes NK cell development, surveillance of MM, and direct as well as ADCC-mediated cytotoxicity against MM targets. Taken together, these data strongly support the concept of targeting AHR as a novel direct anti-MM therapy and anti-MM immunotherapeutic strategy. Disclosures No relevant conflicts of interest to declare.