Browsing by Subject "Therapeutic resistance"

Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer that is characterized by survival signaling through overexpression and/or activation of the epidermal growth factor receptors EGFR/ErbB1 and Her2/ErbB2 and defects in the apoptotic program. The development of therapeutic resistance is a significant barrier to successful treatment in IBC, and thus, strategies targeting the mechanisms that drive drug resistance could prevent or reverse therapeutic resistance, significantly improving patient prognosis. Based on analysis of previously developed models of therapeutic resistant IBC, we hypothesized that apoptotic dysregulation and redox adaptive mechanisms were central to the drug resistant phenotype in IBC cells, and that targeting of these mechanisms could overcome therapeutic resistance. Our objectives to address this hypothesis were: 1. to develop and characterize an isotype-matched IBC cellular model to investigate the mechanisms of acquired therapeutic resistance; 2. to characterize IAP-specific small molecule inhibitors as a means of targeting the mechanism of apoptotic dysregulation in IBC; and 3. to characterize a novel redox modulatory combination as a means of targeting redox adaptive mechanisms in IBC.

Analysis of cell viability, proliferation, and growth parameters, evaluation of protein expression and signaling via western immunoblot, and measurement of reactive oxygen species (ROS), antioxidants, and apoptosis in patient-derived IBC cell lines and isogenic derivatives revealed that resistance to the ErbB1/2 inhibitor lapatinib was protective against other targeted agents and chemotherapeutics. Additionally, reversal of resistance was associated with enhanced ability to accumulate ROS and downregulation of anti-apoptotic and antioxidant proteins. Targeting of resistance mechanisms using small molecule IAP inhibitors and a redox modulatory strategy both effectively induced apoptosis in therapy resistant IBC cells. Together, these results confirm XIAP and the redox adaptive phenotype as promising therapeutic targets for IBC and demonstrate the feasibility of targeting those mechanisms in order to reverse therapeutic resistance.

Targeted therapies rarely yield complete tumor responses, and the residual cancer cells that survive upfront treatment act as a reservoir from which eventual resistant disease emerges. Here, we explore several clinically relevant models of disease resistance, with special attention placed on KRAS-driven colorectal cancer (CRC) and EGFR-driven non-small-cell lung cancer (NSCLC).

First, we note that KRAS mutations drive resistance to diverse targeted therapies, including EGFR inhibitors in colorectal cancer (CRC). Through genetic screens, we unexpectedly find that mutant HRAS, which is rarely found in CRC, is a stronger driver of resistance than mutant KRAS. This difference is ascribed to common codon bias in HRAS, which leads to much higher protein expression, and implies that the inherent poor expression of KRAS due to rare codons must be surmounted during drug resistance. In agreement, we demonstrate that primary resistance to cetuximab is dependent upon both KRAS mutational status and protein expression level, and acquired resistance is often associated with KRASQ61mutations that function even when protein expression is low. Finally, we show that cancer cells upregulate translation to facilitate KRASG12-driven acquired resistance, resulting in hypersensitivity to translational inhibitors. These findings demonstrate that codon bias plays a critical role in KRAS-driven resistance and provide a rationale for targeting translation to overcome resistance.

Next, we demonstrate that targeted therapies induce DNA double strand breaks and consequent, ATM-dependent DNA repair in tumor cells that survive upfront treatment. This DNA damage response, observed in both laboratory models and human patients, is driven by a pathway involving the sub-lethal activation of executioner caspases 3 and 7 and the downstream caspase-activated DNase (CAD). As a consequence, tumor cells that survive upfront treatment harbor a synthetic dependence on ATM, and combined treatment with targeted therapies and a selective ATM kinase inhibitor eradicates these cells, leading to more penetrant and durable responses in in vitro and in vivo models of EGFR-mutant NSCLC. Finally, rare patients with EGFR-mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM show evidence of extended progression-free survival relative to patients lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapy paradigms.

Combined, these studies provide mechanistic-based rationale for pharmacological targeting of tumor-specific processes that may overcome intrinsic and/or acquired resistance states, serving as potential novel therapeutic options for genetically defined subsets of cancer patients.

Apoptotic dysregulation is a hallmark of cancer cells. The inability of cancer cells to undergo apoptosis may lead to therapeutic resistance. Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression and ErbB2 activation. ErbB-targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. Little is known about the molecular mechanisms of therapeutic resistance in IBC cells. We hypothesized that apoptotic dysregulation leads to therapeutic resistance of IBC cells to therapeutic agents, including ErbB-targeting agents. To determine whether apoptotic dysregulation and changes in anti-apoptotic proteins leads to resistance of IBC cells to therapeutic agents, we performed a variety of in vitro-based studies using agents that are used in the clinic to treat IBC patients. The sensitivity of both ErbB2 overexpressing and ErbB1 activated IBC cells to various therapeutic agents was evaluated using various cell death and apoptosis assays, and anti-apoptotic protein expression post-treatment was determined using western blot analysis. The overarching theme observed was that x-linked inhibitor of apoptosis protein (XIAP) expression inversely correlated with sensitivity of cells to therapeutic agents with various mechanisms of action, including TNF-related apoptosis inducing ligand (TRAIL), doxorubicin, cisplatin, paclitaxel, and two ErbB-targeting agents: trastuzumab and a lapatinib-analog (GW583340). Moreover, there was a specific and marked overexpression of XIAP in cells with de novo resistance to trastuzumab and with acquired resistance to GW583340. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. Stable XIAP overexpression using a lentiviral system reversed sensitivity to therapeutic agents (TRAIL and GW583340) in parental IBC cells. Moreover, XIAP downregulation in cells resistant to therapeutic agents (TRAIL, trastuzumab, and GW583340) resulted in decreased viability and increased apoptosis, demonstrating that XIAP is required for survival of cells with resistance to these agents. A novel mechanism of GW583340 oxidative stress-induced mediated apoptosis was identified, and resistant cells had increased antioxidant expression and capability. Interesting, inhibition of XIAP function overcame this increase in antioxidant potential, demonstrating a new function for XIAP in oxidative stress-induced apoptosis. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to therapeutic agents.

Glioblastoma (GBM) is notorious for its immunosuppressive tumor microenvironment (TME). GBM is universally lethal and remains highly refractory to immunotherapy, including immune checkpoint blockade (ICB). Resistance to ICB is a central issue in GBM and is thought to be primarily driven by tumor-imposed immune dysfunction. Here, however, we identify calmodulin-dependent kinase kinase 2 (CaMKK2) as a novel driver of ICB resistance. CaMKK2 is highly expressed in myeloid cells and neurons and is associated with worsened survival in patients with GBM. Using CaMKK2-deficient preclinical murine models, we determine that host CaMKK2 expression reduces survival and promotes ICB resistance in a T cell-dependent manner. Single-cell RNA-sequencing, flow cytometric profiling, and immunofluorescence staining of immune cells in the tumor reveal that CaMKK2 expression is associated with several pro-tumor, ICB resistance-associated immune phenotypes. For instance, CaMKK2 promotes terminal exhaustion in CD8+ T cells and reduces the expansion of effector CD4+ T cells, additionally limiting their tumor penetrance and interactions with myeloid cells. CaMKK2 also maintains myeloid cells in an Apolipoprotein E+, disease-associated microglia-like phenotype, which is associated with ICB resistance. Conversely, CaMKK2 deficiency permits the programming of tumor-associated macrophages (TAMs) to a dendritic cell (DC)-like phenotype that is associated with ICB response. Finally, we determine that it is neuronal CaMKK2 expression, specifically, that is required for maintaining the ICB resistance-associated MHC-IIlow TAM phenotype. Our findings reveal CaMKK2 as a novel contributor to ICB resistance, primarily via non-hematopoietic cells, in GBM and additionally newly identify neurons as a critical driver of pro-tumor immune phenotypes within the GBM TME.

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.