We have previously shown that the d16HER2 splice variant is linked to HER2-positive breast cancer (BC) tumorigenesis, progression and response to Trastuzumab. However, the mechanisms by which d16HER2 contributes to HER2-driven aggressiveness and targeted therapy susceptibility remain uncertain. Here, we report that the d16HER2-positive mammary tumor cell lines MI6 and MI7, derived from spontaneous lesions of d16HER2 transgenic (tg) mice and resembling the aggressive features of primary lesions, are enriched in the expression of Wnt, Notch and epithelial–mesenchymal transition pathways related genes compared with full-length wild-type (WT) HER2-positive cells (WTHER2_1 and WTHER2_2) derived from spontaneous tumors arising in WTHER2 tg mice. MI6 cells exhibited increased resistance to anoikis and significantly higher mammosphere-forming efficiency (MFE) and self-renewal capability than the WTHER2-positive counterpart. Furthermore, d16HER2-positive tumor cells expressed a higher fraction of CD29High/CD24+/SCA1Low cells and displayed greater in vivo tumor engraftment in serial dilution conditions than WTHER2_1 cells. Accordingly, NOTCH inhibitors impaired mammosphere formation only in MI6 cells. A comparative analysis of stemness-related features driven by d16HER2 and WTHER2 in ad hoc engineered human BC cells (MCF7 and T47D) revealed a higher MFE and aldehyde dehydrogenase-positive staining in d16HER2- vs WTHER2-infected cells, sustaining consistent BC-initiating cell enrichment in the human setting. Moreover, marked CD44 expression was found in MCF7_d16 and T47D_d16 cells vs their WTHER2 and Mock counterparts. Clinically, BC cases from two distinct HER2-positive cohorts characterized by high levels of expression of the activated-d16HER2 metagene were significantly enriched in the Notch family and signal transducer genes vs those with low levels of the metagene.

Despite different molecular tumor profiles indicate that human epidermal growth factor receptor 2 (HER2) messenger RNA (mRNA) levels mirror HER2 addiction and trastuzumab benefit in HER2‐positive breast cancer (BC), the identification of noninvasive clinical predictors of trastuzumab sensitivity remains an unmet clinical need. In the current study, we investigated whether intratumor lactate levels reflect HER2 addiction and, in turn, trastuzumab susceptibility. Accordingly, the gene expression profiles of transgenic murine BC cell lines expressing the human d16HER2 variant (HER2‐addicted) or human full‐length HER2 (WTHER2; HER2‐nonaddicted) revealed a significant enrichment of glycolysis‐related gene pathways in HER2‐addicted cells. We studied the metabolic content of 22 human HER2‐positive BC by quantitative nuclear magnetic resonance spectroscopy and found that those cases with higher lactate levels were characterized by higher HER2 transcript levels. Moreover, gene expression analyses of HER2‐positive BC samples from a TCGA data set revealed a significant enrichment in glycolysis‐related pathways in high/HER2‐addicted tumors. These data were confirmed by metabolic analyses of human HER2‐positive BC cell lines with high or low HER2 transcript levels, which revealed significantly more active glycolytic metabolism in high HER2 transcript than in low HER2 transcript cells. Overall, our results provide evidence for noninvasive intratumor lactate detection as a potential metabolic biomarker of HER2 addiction and trastuzumab response suggesting the possibility to use in vivo imaging to assess lactate levels and, in turn, select HER2‐positive BC patients who are more likely to benefit from anti‐HER2 treatments.

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INTRODUCTION: Lenalidomide monotherapy exerts clinical activity in relapsed/refractory Diffuse Large B-cell Lymphoma (DLBCL) with better response rate and progression-free survival being recorded in activated B-cell-like (ABC) rather than germinal center B-cell-like (GCB)-DLBCL. Reasons for such a difference are likely due to different expression of key molecules involved in mediating activity of Lenalidomide, such as Interferon regulatory factor 4(IRF4) and cereblon (CRBN). Evidences supporting the key role of DNA methylation and histone modifications in regulating genome stability and gene expression in DLBCL prompted us to investigate the capacity of Azacytidine in modulating Lenalidomide activity, thereby sensitizing GCB-DLBCL to Lenalidomide and enhancing Lenalidomide efficacy in ABC-DLBCL. METHODS: DLBCL cell lines with ABC (U-2932, RIVA) or GCB (SU-DHL4, SU-DHL6) genotype were used to investigate the effects of Lenalidomide and Azacytidine on cell growth and cell death. Western blotting (WB) and immunofluorescence analysis were used to assess modulating effects of the two-drug combination on molecular determinants of Lenalidomide activity. Additionally, we studied CRBN, IRF4 and CRBN binding proteins expression, such as Ikaros and Aiolos (IKZF1 and IKZF3) by real time polymerase chain reaction (RT-PCR) in response to drug treatment. RESULTS: Graded concentrations of Lenalidomide (0.1-100 µM) inhibited cell proliferation by 20% to 40% and increased cell death up to 30% to 40% in ABC-DLBCL cell lines, whereas had minimal effects on GCB-DLBCL cell lines. Untreated ABC-DLBCL but not GCB-DLBCL consistently showed a high expression of CRBN and IRF4. Upon Lenalidomide treatment (3 days) CRBN was significantly upregulated and IRF4 downregulated in ABC-DLBCL, but not GCB-DLBCL cells. Since DNA methylation regulates gene expression in DLBCL cell lines, we next examined whether Azacytdine could modulate CRBN and IRF4 expression and in turn enhance responsiveness to Lenalidomide. Exposure of both ABC- and GCB-DLBCL cell lines to Azacytidine (up to 72 hours) induced a marked increase of CRBN and IRF4 transcripts; addition of Lenalidomide strongly increased Azacytidine-induced increase of CRBN and significantly downregulated IRF4 expression; the combined treatment induced a marked downregulation of Ikaros and Aiolos protein levels. At the cellular level, the concomitant Azacytidine (10 μM)/Lenalidomide (10 μM) treatment inhibited in a synergistic manner the mean (±SEM) cell growth of both ABC-DLBCL (Lena: -16 ± 4%; AZA: -22 ± 2%; AZA/Lena: -70 ± 1%, P<0.001) and GCB-DLBCL (Lena: -17 ± 3%; AZA: -40 ± 4%; AZA/Lena: -82 ± 2%, P<0.001). Additionally, the two drug exposure was associated with a 3-fold decrease of S phase cells(Lena: 28 ± 2%; AZA: 22 ± 0.8%; AZA/Lena: 9 ± 1%, P<0.001); a marked p21 overexpression, and a 3- to 4-fold cell death increase (P<0.001) in both ABC- and GCB-DLBCL. CONCLUSIONS: Our results indicate that Azacytidine sensitizes GCB-DLBCL to the cytotoxic effects of Lenalidomide and enhances Lenalidomide efficacy against ABC-DLBCL resulting in synergistic anti-proliferative and pro-apoptotic effects in both ABC- and GCB-DLBCL cell lines. Cytotoxicity of the two drug combination is mediated by signaling events involving CRBN upregulation and IRF4 downregulation leading to CRBN-binding proteins downregulation. Azacytidine-dependent activation of CRBN and IRF4 expression allow to hypothesize a methylation-driven regulation of these genes. These results might provide a rationale for clinical studies using Azacytidine and Lenalidomide combination in ABC- and GCB-DLBCL. Disclosures No relevant conflicts of interest to declare.

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