An anti-EGFR antibody-drug conjugate overcomes resistance to HER2-targeted drugs.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved in 2013 to treat HER2+ breast cancer. Despite its efficacy in the clinic, some patients exhibit intrinsic or acquired resistance to such ADC. To characterize mechanisms of resistance to T-DM1, we isolated several HER2+ resistant clones derived from the HCC1954 HER2+ cell line. The isolated clones were different as per their transcriptomic profiles. However, all the T-DM1-resistant clones showed decreased HER2 levels. Yet, the clones were still oncogenically dependent on HER2, as indicated by knock down experiments. The decrease in HER2 expression caused acquired resistance to T-DM1 and to other anti-HER2 therapies. Antibody array analyses showed that the epidermal growth factor receptor (EGFR) was expressed in these T-DM1-resistant HCC1954 clones. Indeed, therapies targeting EGFR, particularly cetuximab-DM1, demonstrated a strong anti-proliferative action on cells with acquired resistance to T-DM1 and HER2 loss. The expression of EGFR in cells resistant to T-DM1 offers the possibility of using therapies directed to this receptor to combat resistance to anti-HER2 drugs and loss of HER2 overexpression.

HER3 in cancer: from the bench to the bedside.

The HER3 protein, that belongs to the ErbB/HER receptor tyrosine kinase (RTK) family, is expressed in several types of tumors. That fact, together with the role of HER3 in promoting cell proliferation, implicate that targeting HER3 may have therapeutic relevance. Furthermore, expression and activation of HER3 has been linked to resistance to drugs that target other HER receptors such as agents that act on EGFR or HER2. In addition, HER3 has been associated to resistance to some chemotherapeutic drugs. Because of those circumstances, efforts to develop and test agents targeting HER3 have been carried out. Two types of agents targeting HER3 have been developed. The most abundant are antibodies or engineered antibody derivatives that specifically recognize the extracellular region of HER3. In addition, the use of aptamers specifically interacting with HER3, vaccines or HER3-targeting siRNAs have also been developed. Here we discuss the state of the art of the preclinical and clinical development of drugs aimed at targeting HER3 with therapeutic purposes.

Therapy-Induced Senescence Enhances the Efficacy of HER2-Targeted Antibody-Drug Conjugates in Breast Cancer.

Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression. SIGNIFICANCE: Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.

Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer.

Cyclin-dependent kinases (CDKs) are a broad family of proteins involved in the cell cycle and transcriptional regulation. In this article, we explore the antitumoral activity of a novel proteolysis-targeting chimera (PROTAC) compound against CDK9. Breast cancer cell lines from different subtypes were used. Transcriptomic mapping of CDKs in breast cancer demonstrated that the expression of CDK9 predicted a detrimental outcome in basal-like tumors (HR = 1.51, CI = 1.08-2.11, p = 0.015) and, particularly, in the luminal B subtype with HER2+ expression (HR = 1.82, CI = 1.17-2.82, p = 0.0069). The novel CDK9 PROTAC, THAL-SNS-032, displayed a profound inhibitory activity in MCF7, T47D, and BT474 cells, with less effect in SKBR3, HCC1569, HCC1954, MDA-MB-231, HS578T, and BT549 cells. The three cell lines with HER2 overexpression and no presence of ER, SKBR3, HCC1569, and HCC1954 displayed an EC50 three times higher compared to ER-positive and dual ER/HER2-positive cell lines. BT474-derived trastuzumab-resistant cell lines displayed a particular sensitivity to THAL-SNS-032. Western blot analyses showed that THAL-SNS-032 caused a decrease in CDK9 levels in BT474, BT474-RH, and BT474-TDM1R cells, and a significant increase in apoptosis. Experiments in animals demonstrated an inverse therapeutic index of THAL-SNS-032, with doses in the nontherapeutic and toxic range. The identified toxicity was mainly due to an on-target off-tumor effect of the compound in the gastrointestinal epithelium. In summary, the potent and efficient antitumoral properties of the CDK9 PROTAC THAL-SNS-032 opens the possibility of using this type of compound in breast cancer only if specifically delivered to cancer cells, particularly in ER/HER2-positive and HER2-resistant tumors.

JKST6, a novel multikinase modulator of the BCR-ABL1/STAT5 signaling pathway that potentiates direct BCR-ABL1 inhibition and overcomes imatinib resistance in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.

Generation of Antibody-Drug Conjugate Resistant Models.

In the last 20 years, antibody-drug conjugates (ADCs) have been incorporated into the oncology clinic as treatments for several types of cancer. So far, the Food and Drug Administration (FDA) has approved 11 ADCs and other ADCs are in the late stages of clinical development. Despite the efficacy of this type of drug, the tumors of some patients may result in resistance to ADCs. Due to this, it is essential not only to comprehend resistance mechanisms but also to develop strategies to overcome resistance to ADCs. To reach these goals, the generation and use of preclinical models to study those mechanisms of resistance are critical. Some cells or patient tumors may result in primary resistance to the action of an ADC, even if they express the antigen against which the ADC is directed. Isolated primary tumoral cells, cell lines, or patient explants (patient-derived xenografts) with these characteristics can be used to study primary resistance. The most common method to generate models of secondary resistance is to treat cancer cell lines or tumors with an ADC. Two strategies, either continuous treatment with the ADC or intermittent treatment, have successfully been used to develop those resistance models.

Altered proTGFα/cleaved TGFα ratios offer new therapeutic strategies in renal carcinoma.

BACKGROUND: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer. METHODS: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα). RESULTS: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed. CONCLUSIONS: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein.

Novel ADCs and Strategies to Overcome Resistance to Anti-HER2 ADCs.

During recent years, a number of new compounds against HER2 have reached clinics, improving the prognosis and quality of life of HER2-positive breast cancer patients. Nonetheless, resistance to standard-of-care drugs has motivated the development of novel agents, such as new antibody-drug conjugates (ADCs). The latter are a group of drugs that benefit from the potency of cytotoxic agents whose action is specifically guided to the tumor by the target-specific antibody. Two anti-HER2 ADCs have reached the clinic: trastuzumab-emtansine and, more recently, trastuzumab-deruxtecan. In addition, several other HER2-targeted ADCs are in preclinical or clinical development, some of them with promising signs of activity. In the present review, the structure, mechanism of action, and potential resistance to all these ADCs will be described. Specific attention will be given to discussing novel strategies to circumvent resistance to ADCs.

HER3 targeting with an antibody-drug conjugate bypasses resistance to anti-HER2 therapies.

Despite impressive clinical benefit obtained with anti-HER2-targeted therapies, in advances stages, especially in the metastatic setting, HER2-positive tumors remain incurable. Therefore, it is important to develop novel strategies to fight these tumors, especially when they become resistant to available therapies. We show here that the anti-HER3 antibody-drug conjugate EV20/MMAF exerted potent anti-tumoral properties against several models of primary resistance and secondary resistance to common anti-HER2 available therapies, including trastuzumab, lapatinib, neratinib, and trastuzumab-emtansine. HER3 was expressed in these HER2+ breast cancer cells and knockdown experiments demonstrated that HER3 expression was required for the action of EV20/MMAF. In mice injected with trastuzumab-resistant HER2+ cells, a single dose of EV20/MMAF caused complete and long-lasting tumor regression. Mechanistically, EV20/MMAF bound to cell surface HER3 and became internalized to the lysosomes. Treatment with EV20/MMAF caused cell cycle arrest in mitosis and promoted cell death through mitotic catastrophe. These findings encourage the clinical testing of EV20/MMAF for several indications in the HER2+ cancer clinic, including situations in which HER2+ tumors become refractory to approved anti-HER2 therapies.

Adaptive resistance to trastuzumab impairs response to neratinib and lapatinib through deregulation of cell death mechanisms.

Small molecule inhibitors (TKIs) of HER2 have demonstrated clinical benefit in HER2-positive breast tumors. One of them, lapatinib, is used once advanced tumors become refractory to the HER2 antibody trastuzumab. Another one, neratinib, has shown benefit in high-risk early-stage breast cancer after trastuzumab-based therapies. A common characteristic is that patients are formerly treated with trastuzumab. We have explored whether trastuzumab previous therapy affects its antitumoral action. Long time exposure of the HER2+ cell line BT474 to trastuzumab resulted in trastuzumab-insensitive cells (BTRH cells). While treatment of wild type BT474 cells with lapatinib or neratinib resulted in decreased viability, BTRH cells were resistant to the action of these TKIs. Analogous results were obtained using trastuzumab-resistant cells derived from a PDX. Functional transcriptomic analyses and biochemical studies demonstrated that the TKIs caused DNA damage and apoptosis in wild type cells, but not in BTRH. Moreover, previous treatment with trastuzumab impairs response to small TKIs, by eliminating their proapoptotic action. Moreover, actioning on the apoptotic machinery using a chemical library of proapoptotic compounds led to the identification of clinical-stage drugs that may be used to fight trastuzumab-TKI resistance.