Immunotherapy in Breast Cancer: When, How, and What Challenges?

Breast Cancer (BC) is the second most frequent cause of cancer death among women worldwide and, although there have been significant advances in BC therapies, a significant percentage of patients develop metastasis and disease recurrence. Since BC was demonstrated to be an immunogenic tumor, immunotherapy has broken through as a significant therapy strategy against BC. Over the years, immunotherapy has improved the survival rate of HER2+ BC patients due to the approval of some monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab and, recently, Margetuximab, along with the antibody-drug conjugates (ADC) Trastuzumab-Emtansine (T-DM1) and Trastuzumab Deruxtecan. Immune checkpoint inhibitors (ICI) showed promising efficacy in triple-negative breast cancer (TNBC) treatment, namely Atezolizumab and Pembrolizumab. Despite the success of immunotherapy, some patients do not respond to immunotherapy or those who respond to the treatment relapse or progress. The main causes of these adverse events are the complex, intrinsic or extrinsic resistance mechanisms. In this review, we address the different immunotherapy approaches approved for BC and some of the mechanisms responsible for resistance to immunotherapy.

Next-Generation STAT3 Inhibitors As Targeted Therapeutics in Chronic Myeloid Leukemia.

Abstract 2445 Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors. Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705. We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p<0.02) and 27.5% (p<0.004) of controls, respectively. In contrast, SF1–066 did not synergize with imatinib in parental K562 and AR230 cells that lack constitutive pSTAT3Y705 (p>0.05). Next, CD34+ cells from newly diagnosed CML patients (n=4) were cultured for 96 hours in the presence of CM and treated with 2.5 μM imatinib, 10 μM SF1–066 or both. Equal numbers of cells were then plated in colony forming assays. Imatinib combined with SF1–066 reduced colony formation to 54.8% (p<0.002) of controls treated with imatinib. Given that dual treatment reduces but does not completely suppress colony formation, we reasoned that some STAT3 signaling must persist in the presence of SF1–066. Thus, we developed next-generation STAT3 inhibitors with increased activity compared to SF1–066. Computational modeling informed the design and synthesis of a second-generation, SF1–066-based library. We evaluated compounds with shared molecular functionalities using AR230R cells expressing a luciferase reporter containing sequential STAT3 sis-inducible elements (AR230R-SIE). At 10 μM, we found two inhibitors, BP2–047 and BP3–163, that reduced luminescence by 69.2% (p<3.6×10−6) and 59.7% (p<8.6×10−6), respectively, compared to controls (n=3). However, the compounds also reduced luminescence in AR230R cells expressing a scrambled luciferase reporter (AR230R-NEG). This screen provided information on the structure-activity relationships of the compounds and was used to generate 64 more candidate STAT3 inhibitors. To account for the reduced luminescence in AR230R-NEG cells and analyze for more potent inhibitors, we performed the luciferase assay with candidate inhibitors at 5 μM. We identified BP5–087 and BP5–088, each with increased potency and STAT3 selectivity compared to SF1–066. These compounds decreased luminescence in AR230R-SIE cells by 36.1% (p<2.6×10−7) and 25.5% (p<6.1×10−10), respectively (n=3), with minimal effects on AR230R-NEG cells (p>0.05). Confirmation of STAT3 binding was obtained using fluorescence polarization assays, in which the EC50 values of BP5–087 and BP5–088 measured 8.5 μM and 4.6 μM, respectively. Studies with mouse and human liver microsomes also revealed that BP5–087 and BP5–088 exhibit enhanced metabolic stability compared to SF1–066. Interestingly, treatment with BP5–087 or BP5–088 (both at 1 and 5 μM) in CML CD34+ progenitors grown in CM showed increased cytoplasmic accumulation of pSTAT3Y705 compared to controls. Thus, we identified BP5–087 and BP5–088 as two of the most potent small-molecule binders of STAT3 reported. These compounds are promising frontrunners toward new therapies for TKI-resistant CML and other diseases in which STAT3 activation drives malignant phenotypes. Disclosures: No relevant conflicts of interest to declare.

Intrinsic and Extrinsic Survival Signals Converge on STAT3 As a Critical Mediator of BCR-ABL-Independent Tyrosine Kinase Inhibitor Resistance,

Abstract 3742 In Philadelphia chromosome positive leukemia, mutations in the BCR-ABL kinase domain are a well established mechanism of resistance to tyrosine kinase inhibitors (TKIs), but fail to explain many cases of primary and acquired resistance. Extrinsic survival signals from the bone marrow microenvironment can protect CML cells from the effects of TKIs in a STAT3-dependent manner. To define the role of pSTAT3 in extrinsic versus intrinsic TKI resistance, we used BCR-ABL+ cell lines exhibiting ‘extrinsic resistance' through culture in conditioned media (CM) from human HS-5 bone marrow stromal cells (Re), or derivative lines expressing native BCR-ABL with ‘intrinsic resistance' adapted for growth in the presence of 1.0–2.5 μM imatinib (Ri). Immunoblot analysis showed near-complete suppression of BCR-ABL tyrosine kinase activity upon exposure to imatinib in all Re and Ri cells tested, suggesting BCR-ABL-independent resistance in both cases. K562-Ri and AR230-Ri cells proliferating in 1.0 μM imatinib showed upregulation of pAKTS473, pJAK2Y1007/1008, pSTAT3, and pSTAT5 compared to sensitive cells under the same conditions. When imatinib was increased (2.5 μM), pJAK2Y1007/1008 and pSTAT5 were markedly reduced, suggesting regulation by BCR-ABL kinase activity. In contrast, levels of pSTAT3 and pAKTS473 were further increased. Similar to Ri cells, extrinsically resistant cells (K562-Re, LAMA84-Re, KBM5-Re) showed increased pSTAT3 when cultured in HS-5 CM; however, upregulation was not observed for pAKTS473. Of key importance, pSTAT3 levels were also increased by HS-5 CM in CD34+ cells from newly diagnosed CML patients. To further investigate the role of pSTAT3 in TKI resistance, we used lentiviral shRNA to knockdown STAT3 (shSTAT3) in sensitive and resistant BCR-ABL+ cells. Cells were kept in culture for 24 hours with and without 1.0 μM imatinib in regular medium or CM, followed by trypan blue exclusion and/or plating in semisolid medium. Compared to cells expressing a scrambled control (shSCR), shSTAT3 reduced the in vitro growth of intrinsically resistant K562-Ri and AR230-Ri cells by 54.6% and 33.3% in the presence of imatinib (1.0 μM), respectively. Consistent with these observations, shSTAT3 impaired the clonogenic potential of K562-Ri cells by 65.0% following culture in 1.0 μM imatinib, and similar results were obtained for AR230-Ri cells, indicating that STAT3 plays a functional role in mediating intrinsic TKI resistance in multiple BCR-ABL-expressing cell lines. In extrinsic resistance, 3–12 hours of culture with CM from HS-5 stromal cells enhanced the clonogenic potential of K562-Re and LAMA84-Re cells by 25–40% in the presence of imatinib compared to controls cultured in regular medium. However, the protective effects of CM were abrogated by introduction of shSTAT3. In contrast, shSTAT3 had no effect on colony formation following growth in regular medium, consistent with the low level of pSTAT3 in parental K562 and LAMA-84 cells. These data suggest that STAT3 activation is a prominent feature of both extrinsic and intrinsic BCR-ABL-independent imatinib resistance. Additionally, preliminary experiments implicate pSTAT3 in intrinsic BCR-ABL-independent resistance to the third-generation TKI, ponatinib (AP24534), suggesting that activated STAT3 may have a role in resistance to other TKIs. Altogether, our data suggest that intrinsic and extrinsic pathways converge on STAT3 as a critical mediator of BCR-ABL-independent TKI resistance, and implicate this pathway as a potential therapeutic target for the treatment of patients with TKI resistance despite BCR-ABL inactivation. We posit that microenvironmental cues activate pathways that initially support survival of leukemia cells despite BCR-ABL inhibition, and that overt resistance develops when these cells establish an intrinsic mechanism to maintain activation of the same pathways. Disclosures: No relevant conflicts of interest to declare.