Force-sensing treadmill gait analysis system can detect gait abnormalities in haemophilia patients without arthropathy.

BACKGROUND: Joint damage in patients with haemophilia (PwH) is commonly assessed by imaging, but few reports have described how structural changes in joints, for example, haemophilic arthropathy (HA)-affect gait ability. OBJECTIVES: We evaluated gait function among PwH with HA, PwH without HA, and people without haemophilia (non-PwH) using a Zebris FDM-T treadmill (FDM-T), an easy-to-use gait assessment instrument with a force sensor matrix. METHODS: The following gait parameters were collected: centre of pressure trajectory intersection (COPi) anterior/posterior variability, COPi lateral variability, COPi anterior/posterior symmetry, COPi lateral symmetry, single-limb support line (SLSL) length, and SLSL variability. Participants walked at their typical gait speed. The physical function of the PwH was assessed by the Hemophilia Joint Health Score (HJHS). Parameters were compared among the three groups. RESULTS: Twelve PwH with HA, 28 PwH without HA, and 12 non-PwH were enrolled. Gait speed significantly differed between groups (non-PwH, 3.1 ± 0.7; PwH without HA, 2.0 ± 0.7; PwH with HA; 1.5 ± 0.4). The COPi anterior/posterior variability, COPi lateral variability, SLSL length, and SLSL variability were greater in the PwH groups than in the non-PwH group. The COPi lateral symmetry differed between PwH with HA and the other groups. The HJHS was not correlated with gait parameters among PwH with HA. CONCLUSIONS: Gait parameters and speed were abnormal in both PwH with HA and PwH without HA. The FDM-T can be used to identify early stages of physical dysfunction that cannot be detected by conventional functional assessments such as the HJHS.

The use of infrared thermography for non-invasive detection of bleeding and musculoskeletal abnormalities in patients with hemophilia: an observational study.

BACKGROUND: In patients with hemophilia (PwH), bleeding often occurs in joints and muscles, and early detection of hemorrhage is important to prevent the onset and progression of mobility impairment. Complex-Image analysis such as ultrasonography, computed tomography, and magnetic resonance imaging are used to detect bleeding. On the other hand, no simple and rapid method to detect the active bleeding has been reported. Local inflammatory responses occur when blood leaks from damaged vessels, and the temperature at the site of active bleeding could be expected to increase in these circumstances, leading to an increase in surrounding skin temperature. Therefore, the purpose of this study was to investigate whether the measurement of skin temperature using infrared thermography (IRT) can be used as a diagnostic aid to detect active bleeding. METHODS: Fifteen PwH (from 6 to 82 years old) complaining of discomfort such as pain were examined. Thermal images were obtained simultaneously at the affected sides and comparable unaffected sides. The average skin temperature of the affected side and of the unaffected side were measured. The temperature differences were calculated by subtracting the average skin temperature at the unaffected side from the affected side. RESULTS: In eleven cases with active bleeding, the skin temperature at the affected side was more than 0.3 °C higher (0.3 °C to 1.4 °C) compared to the unaffected side. In two cases without active bleeding, there were no significant differences in skin temperature between the affected and unaffected sides. In two cases with previous rib or thumb bone fracture, the skin temperature at the affected side was 0.3 °C or 0.4 °C lower than that of the unaffected side, respectively. In two cases with active bleeding in which longitudinal evaluation was conducted, the difference in skin temperature decreased after hemostatic treatment. CONCLUSION: The analysis of skin temperature deference using IRT was a useful supportive tool to readily assess musculoskeletal abnormalities and bleeding in PwH as well as to determine the success of the hemostatic treatment.

The molecular rationale for the combination of polatuzumab vedotin plus rituximab in diffuse large B-cell lymphoma.

Polatuzumab vedotin (Pola) is an antibody-drug conjugate that targets the B-cell antigen CD79b and delivers monomethyl auristatin E (MMAE). It is approved in combination with bendamustine and rituximab (Rit) for relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Understanding the molecular basis of Pola combination therapy with Rit, the key component for the treatment of DLBCL, is important to establish the effective treatment strategies against r/r DLBCL. Here, we examined the rationale for the combination of Pola with Rit using Pola-refractory cells. We found that treatment with Pola increased CD20 expression and sensitivity to Rit-induced complement-dependent cytotoxicity (CDC) in several Pola-refractory cells. Pola treatment increased phosphorylation of AKT and ERK and both AKT- and MEK-specific inhibitors attenuated the Pola-induced increase of CD20 and CDC sensitivity, suggesting that these phosphorylation events were required for this combination efficacy. It was revealed that anti-CD79b antibody increased the phosphorylation of AKT but inhibited the phosphorylation of ERK. In contrast, MMAE potentiated phosphorylation of ERK but slightly attenuated the phosphorylation of AKT. Pola also increased CD20 expression on Pola-refractory xenografted tumours and significantly enhanced antitumour activity in combination with Rit. In conclusion, these results could provide a novel rationale for the combination of Pola plus Rit.

Sensitivity of eight types of ALK fusion variant to alectinib in ALK-transformed cells.

Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non-small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a <3.6-fold difference in responsiveness. In conclusion, these eight ALK variants had similar sensitivity to alectinib in vitro, indicating that it may not be possible to predict the response to alectinib just by determination of the ALK variant type in ALK fusion-positive NSCLCs.

Resistance to obinutuzumab-induced antibody-dependent cellular cytotoxicity caused by abnormal Fas signaling is overcome by combination therapies.

BACKGROUND: Obinutuzumab, a Type II anti-CD20 antibody, is used to treat follicular lymphoma. A major mode of action of obinutuzumab is antibody-dependent cellular cytotoxicity (ADCC). Knowledge of the mechanisms of resistance to obinutuzumab is important for the development of next-line strategies to follow obinutuzumab-containing therapy, including obinutuzumab retreatment. Unfortunately, the mechanisms by which tumor cells acquire resistance to ADCC are still poorly understood. To address this, we examined the mechanisms of resistance to obinutuzumab-induced ADCC and the combination efficacy of obinutuzumab and clinically available agents in the established resistant cells. METHODS AND RESULTS: We established cells resistant to obinutuzumab-induced ADCC using the non-Hodgkin lymphoma cell line RL and examined their mechanisms of resistance and the combination efficacy of obinutuzumab and clinically available agents. Comprehensive analysis by RNA sequencing of resistance mechanisms revealed that abnormal Fas signaling decreased sensitivity to ADCC in resistant clones. Combination treatment with prednisolone, a component of CHOP and CVP, was found to enhance ADCC sensitivity of RL cells and resistant clones and to significantly suppress tumor growth in xenograft models. Treatment with prednisolone upregulated expression of CD20 and an apoptosis-inducing protein BIM, which might augment perforin/granzyme B-mediated cell death. Furthermore, pretreatment of the effector cells with bendamustine enhanced ADCC activity, and treatment with obinutuzumab plus bendamustine showed significant antitumor efficacy in xenograft models. It was speculated that bendamustine upregulates ADCC activity by potentiating granules-mediated cell killing. CONCLUSIONS: Our study revealed a novel mechanism underlying obinutuzumab-induced ADCC resistance and indicated that ADCC resistance could be overcome by combining obinutuzumab with prednisolone or bendamustine. This study provides a scientific rationale for obinutuzumab-retreatment in combination with clinically available chemotherapeutic agents for obinutuzumab resistant follicular lymphoma.

Trastuzumab in combination with paclitaxel enhances antitumor activity by promoting apoptosis in human epidermal growth factor receptor 2-positive trastuzumab-resistant gastric cancer xenograft models.

Trastuzumab, a humanized anti-human epidermal growth factor receptor 2 antibody drug, is the first-line therapy for human epidermal growth factor receptor 2-positive breast and gastric cancer. For breast cancer, the benefit of continuous treatment with trastuzumab after it becomes refractory to first-line therapy has been demonstrated. However, it is unclear whether trastuzumab can show similar efficacy as a second-line treatment for gastric cancer. Here, we report that trastuzumab in combination with paclitaxel exhibits increased antitumor efficacy even for trastuzumab-resistant xenografted tumors. We derived the trastuzumab-resistant models from previously established human epidermal growth factor receptor 2-positive gastric cancer patient-derived cells. Human epidermal growth factor receptor 2 expression, PIK3CA mutation, and phosphatase and tensin homolog expression in these resistant models was equivalent to those in the trastuzumab-sensitive parental model, whereas cyclin-dependent kinase inhibitors, such as p16, p15, and p21, were downregulated. Trastuzumab in combination with paclitaxel enhanced antitumor activity in both the sensitive and resistant models. In the trastuzumab-sensitive model, the combination of trastuzumab and paclitaxel resulted in suppression of the AKT-p27-retinoblastoma protein pathway and induction of apoptosis. Although this combination did not suppress retinoblastoma protein phosphorylation in the trastuzumab-resistant model, it did markedly decrease epidermal growth factor receptor and human epidermal growth factor receptor 2 phosphorylation and further enhance paclitaxel-mediated apoptosis. These results suggested that trastuzumab in combination with paclitaxel can still exert more potent antitumor efficacy than each agent alone in trastuzumab-resistant models, providing evidence that trastuzumab remains beneficial in the treatment of trastuzumab-resistant tumors.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion.

BACKGROUND: Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. METHODS: In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription-PCR, respectively. RESULTS: Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion-positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion-negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. CONCLUSIONS: This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

Psychosocial functioning is correlated with activation in the anterior cingulate cortex and left lateral prefrontal cortex during a verbal fluency task in euthymic bipolar disorder: a preliminary fMRI study.

AIM: Cognitive impairment may account for functional and occupational disability in patients with bipolar disorder even during periods of euthymia. While imaging suggests structural, neurochemical, and functional abnormalities in bipolar disorder patients, the pathophysiology of these deficits has not been elucidated. It was hypothesized that euthymic bipolar patients would have different cortical activation during a verbal fluency task compared to healthy controls, and that psychosocial functioning would be associated with prefrontal cortical activation during the task in the bipolar group. METHODS: Ten euthymic bipolar patients and 10 healthy control participants (matched for age, gender, and years of education) underwent functional magnetic resonance imaging (fMRI) during a verbal fluency task, tapping task and visual task. Correlational analysis between the fMRI brain activation and clinical variables of the participants, including Global Assessment of Functioning (GAF) score, was performed. RESULTS: Compared to the controls, euthymic bipolar patients had significantly greater activation in the bilateral precuneus with similar behavioral performance during the verbal fluency task. There were no significant differences between the groups for the visual task or the simple motor task. Activation in both the left anterior cingulate cortex (ACC) and the left dorsolateral prefrontal cortex (PFC) were significantly positively correlated with GAF score in the euthymic bipolar patients. CONCLUSION: Both the ACC and lateral PFC regions are components of a neural network that plays a critical role in psychosocial functioning, and are often found to be affected in bipolar patients.

Efficacy of retreatment with polatuzumab vedotin in combination with rituximab in polatuzumab vedotin-resistant DLBCL models.

Polatuzumab vedotin (Pola) was approved for first-line and relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) in many countries. This means that retreatment with Pola for r/r DLBCL could be considered after first-line Pola treatment; however, there is currently no evidence on the effectiveness of Pola-retreatment. To address this, we established two Pola-resistant cells from DLBCL cells (SU-DHL-4 and STR-428) and evaluated the combination efficacy of Pola plus rituximab (Rit), the key component of DLBCL therapy. MDR1 overexpression and decreased Bim expression were suggested to be the resistant mechanisms to Pola in Pola-resistant SU-DHL-4 and Pola-resistant STR-428, respectively. In these cells, Pola significantly increased Rit-induced CDC sensitivity either with increased MAC formation or reduced Mcl-1 expression. Additionally, treatment with Pola + Rit significantly enhanced antitumor activity in Pola-resistant STR-428 xenograft mouse models. Based on these results, Pola + Rit retreatment could have preserved efficacy because of the effect of Pola on sensitizing cells to Rit.

FGFR blockade inhibits targeted therapy-tolerant persister in basal FGFR1- and FGF2-high cancers with driver oncogenes.

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced anaplastic lymphoma kinase (ALK) fusion-positive DTP cell's survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and fibroblast growth factor 2 (FGF2)-high protein expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs increased in patients with ALK fusion-positive non-small cell lung cancer with FGFR1- and FGF2-high mRNA expression at baseline. The combination of FGFR and targeted TKIs enhanced cell growth inhibition and apoptosis induction in basal FGFR1- and FGF2-high protein expressing cells with ALK-rearranged and epidermal growth factor receptor (EGFR)-mutated NSCLC, human epidermal growth factor receptor 2 (HER2)-amplified breast cancer, or v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma by preventing compensatory extracellular signal-regulated kinase (ERK) reactivation. These results suggest that a targeted TKI-induced DTP state results from an oncogenic switch from activated oncogenic driver signaling to the FGFR1 pathway in basal FGFR1- and FGF2-high expressing cancers and initial dual blockade of FGFR and driver oncogenes based on FGFR1 and FGF2 expression levels at baseline is a potent treatment strategy to prevent acquired drug resistance to targeted TKIs through DTP cells regardless of types of driver oncogenes.

Coadministration with bendamustine restores the antitumor activity of obinutuzumab in obinutuzumab-resistant tumors.

BACKGROUND: The glycoengineered, humanized anti-CD20 antibody obinutuzumab is indicated for previously untreated or relapsed/refractory CD20-positive follicular lymphoma (FL). However, the effectiveness of obinutuzumab retreatment in relapsed/refractory FL after prior obinutuzumab-containing therapy is unclear. To address this issue, we investigated the antitumor activity of obinutuzumab plus bendamustine in obinutuzumab-resistant tumors established from a human non-Hodgkin lymphoma xenograft model. MATERIALS AND METHODS: Obinutuzumab-resistant tumors (SU-DHL-4-OR-18-8) were established from an SU-DHL-4 xenograft model by repeated administration of obinutuzumab. Antitumor activity was evaluated based on tumor volume after treatment with obinutuzumab on Day 1, 8, and 15 and/or bendamustine on Day 1 and 2. Intratumoral natural killer (NK) cells/macrophages were evaluated by immunohistochemistry and flow cytometry. RESULTS: In SU-DHL-4-OR-18-8 xenografted tumors, intratumoral NK cells/macrophages after obinutuzumab treatment were significantly decreased compared with parent tumors on Day 4. The endoplasmic reticulum stress sensor phospho-IRE1 was also decreased. In SU-DHL-4-OR-18-8 tumors, bendamustine treatment increased phospho-IRE1 on Day 4 and intratumor NK cells/macrophages on Day 10. Obinutuzumab combined with bendamustine significantly increased antitumor activity compared with each single agent on Day 29, with an increase in chemoattractant CCL6 expression on Day 10. CONCLUSIONS: Coadministration of bendamustine in obinutuzumab retreatment may be effective against obinutuzumab-resistant tumors.

Identification of a checkpoint modulator with synthetic lethality to p53 mutants.

The G(2) checkpoint is an indispensable pathway for cancers lacking p53 function, for delaying cell cycle progression, and for completing DNA repair. Therefore, disruption of this pathway is expected to offer selective therapy for these highly prevalent cancers. The aim of this study was to identify an inhibitor of the G(2) checkpoint including the ataxia-telangiectasia-mutated and Rad3-related checkpoint kinase 1 pathway that selectively suppresses the growth of p53-deficient cells. To obtain molecules with a novel mechanism of action, we constructed a high-throughput screening system that detected abrogation of the G(2) checkpoint in X-irradiated HT-29 cells. The screening resulted in identification of a guanidine analog, CBP-93872 that dose dependently inhibited the G(2) checkpoint induced by DNA damage. Interestingly, CBP-93872 directly suppressed the growth of p53-mutated cancer cell lines with wild-type CDKN2A by eliciting G(1) arrest, but not CDKN2A-deleted and/or wild-type p53 lines. CBP-93872 decreased phospho-cdc2 Y15 by inhibiting phosphorylation of Chk1, but did not suppress phospho-Chk2 or the kinase activities of either Chk1 or Chk2 in cellular or cell-free assays. These results suggest that a checkpoint modulator through suppression of Chk1 phosphorylation provides synthetic lethality to p53-deficient cells.

Design and synthesis of novel allosteric MEK inhibitor CH4987655 as an orally available anticancer agent.

The MAP kinase pathway is one of the most important pathways involved in cell proliferation and differentiation, and its components are promising targets for antitumor drugs. Design and synthesis of a novel MEK inhibitor, based on the 3D-structural information of the target enzyme, and then multidimensional optimization including metabolic stability, physicochemical properties and safety profiles were effectively performed and led to the identification of a clinical candidate for an orally available potent MEK inhibitor, CH4987655, possessing a unique 3-oxo-oxazinane ring structure at the 5-position of the benzamide core structure. CH4987655 exhibits slow dissociation from the MEK enzyme, remarkable in vivo antitumor efficacy both in mono- and combination therapy, desirable metabolic stability, and insignificant MEK inhibition in mouse brain, implying few CNS-related side effects in human. An excellent PK profile and clear target inhibition in PBMC were demonstrated in a healthy volunteer clinical study.

Obinutuzumab in Combination with Chemotherapy Enhances Direct Cell Death in CD20-Positive Obinutuzumab-resistant Non-Hodgkin Lymphoma Cells.

Follicular lymphoma commonly recurs and is difficult to cure. Obinutuzumab is a humanized glycoengineered type II anti-CD20 antibody with a mode of action that includes induction of antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and direct cell death. There is no evidence on the effectiveness of retreatment with obinutuzumab in patients with prior obinutuzumab treatment. Using obinutuzumab-induced direct-cell-death-resistant cells, we investigated the efficacy of obinutuzumab retreatment in combination with chemotherapeutic agents used in follicular lymphoma treatment. Human non-Hodgkin lymphoma SU-DHL-4 cells were sustainably exposed to obinutuzumab in vitro, and 17 resistant clones expressing CD20 and showing 100-fold higher IC50 of obinutuzumab than parental cells were established. The growth inhibition effect of obinutuzumab in combination with bendamustine, 4-hydroperoxy-cyclophosphamide, doxorubicin, vincristine, or prednisolone was estimated using an interaction index based on the Bliss independence model. For each clone, there were various combinations of obinutuzumab and chemotherapeutic agents that showed supra-additive effects. Obinutuzumab combined with doxorubicin enhanced caspase-dependent apoptosis and growth inhibition effect. Obinutuzumab combined with prednisolone enhanced DNA fragmentation and G0-G1 arrest. These combinations also had an antitumor effect in mouse xenograft models. Our results indicate that retreatment with obinutuzumab, when it is combined with chemotherapeutic agents, is effective in the CD20-positive obinutuzumab-induced direct-cell-death-resistant cells.

Detailed analysis of anti-emicizumab antibody decreasing drug efficacy, using plasma samples from a patient with hemophilia A.

BACKGROUND: Emicizumab is a humanized bispecific monoclonal antibody that bridges activated factor IX (FIXa) and factor X (FX) to mimic the function of factor VIII (FVIII). It suppresses the bleeding tendency in hemophilia A patients with or without FVIII inhibitors. A case of an adult FVIII inhibitor-positive hemophilia A patient in whom treatment with emicizumab was discontinued owing to the repeated bleeding events and prolonged activated partial thromboplastin time. OBJECTIVE: To analyze the mechanisms of decreased efficacy of emicizumab. METHODS: Residual plasma samples were used to measure the following: emicizumab concentration in plasma, measured by enzyme-linked immunosorbent assay; titer of anti-drug antibody (ADA) against emicizumab, measured by electrochemiluminescence; and neutralizing activity against emicizumab, measured by Bethesda method modified by using emicizumab-spiked FVIII-deficient plasma. RESULTS: At week 31, emicizumab concentration was 15.0 µg/ml, and ADAs were measured as positive. Emicizumab concentration continued to decrease until emicizumab discontinuation point at week 49, and after week 50, emicizumab concentrations were below the limitation of quantification. The ADA titer increased transiently from week 31, even past the emicizumab discontinuation point at week 49. The ADA titer then gradually decreased until the last sampling point at week 93. Neutralizing activity against emicizumab was detected after emicizumab discontinuation. Epitope analysis showed that the ADAs recognize the anti-FIXa and anti-FX Fab arms of emicizumab, but not the Fc region. CONCLUSION: The appearance of ADAs with emicizumab-neutralizing activity and potential to accelerate emicizumab clearance decreased the efficacy of emicizumab.

Enhanced antitumor effect of alectinib in combination with cyclin-dependent kinase 4/6 inhibitor against RET-fusion-positive non-small cell lung cancer cells.

Rearranged during transfection (RET) fusion-positive non-small cell lung cancer (NSCLC) accounts for 1% of lung adenocarcinoma. Although small molecule agents with RET kinase inhibitory activity such as alectinib, vandetanib, and cabozantinib have been clinically evaluated in RET-fusion-positive NSCLC, an effective monotherapy regimen has not been established. We explored agents to use in combination with alectinib to enhance the antitumor effect of alectinib against RET-fusion cells. Cell proliferation under co-treatment with alectinib plus each of six chemotherapeutic agents or six molecularly targeted agents was evaluated in vitro. The combination effect was analyzed by IC50 isobologram and combination index using LC-2/ad and Ba/F3-KIF5B-RET cells. The in vivo combination effect was investigated in a Ba/F3-KIF5B-RET xenograft model. The phosphorylation levels of proteins regulating proliferation were measured by immunoblotting. Palbociclib, a CDK4/6 inhibitor, showed the greatest synergy against LC-2/ad cells in the isobologram analysis and combination index. This synergistic effect was also observed against Ba/F3-KIF5B-RET cells. Another CDK4/6 inhibitor, abemaciclib, also showed a synergistic effect. In vivo, the combination of alectinib plus palbociclib showed a more enhanced antitumor effect than each single agent in a mouse xenograft model with transplanted Ba/F3-KIF5B-RET cells. This combination suppressed the phosphorylation of S6 and Rb more intensely than did either single agent in both LC-2/ad and Ba/F3-KIF5B-RET cell lines, both in vitro and in vivo. Combination therapy with alectinib plus the CDK4/6 inhibitor enhanced the antitumor effect against RET-fusion-positive cells in vitro and in vivo.

Combination efficacy of pertuzumab and trastuzumab for trastuzumab emtansine-resistant cells exhibiting attenuated lysosomal trafficking or efflux pumps upregulation.

PURPOSE: Trastuzumab emtansine (T-DM1) is the standard treatment in the current second-line therapy of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. However, a useful therapy after T-DM1 resistance has not been established. In this study, we established two different HER2-positive T-DM1-resistant cancer cells and evaluated the antitumor effect of trastuzumab in combination with pertuzumab (TRAS + PER). METHODS: Single-cell-cloned OE19 and BT-474 cells were cultured with increasing concentrations of T-DM1 to generate T-DM1-resistant OE19bTDR and BT-474bTDR cells, respectively. HER2 expression was assessed by immunohistochemistry. Multidrug resistance proteins (MDR1 and MRP1) were evaluated by real-time polymerase chain reaction and western blotting. Intracellular trafficking of T-DM1 was examined by flow cytometry and immunofluorescence staining. Efficacy of TRAS + PER was evaluated by cell proliferation assay, HER3 and AKT phosphorylation, caspase 3/7 activity, and antitumor activity. RESULTS: HER2 expression of both resistant cells was equivalent to that of the parent cells. Overexpression of MDR1 and MRP1 was observed and affected the T-DM1 sensitivity in the OE19bTDR cells. Abnormal localization of T-DM1 into the lysosomes was observed in the BT-474bTDR cells. In BT-474bTDR cells, TRAS + PER inhibited the phosphorylation of AKT involved in HER2-HER3 signaling, and apoptosis induction and cell proliferation inhibition were significantly higher with TRAS + PER than with the individual drugs. TRAS + PER significantly suppressed tumor growth in the OE19bTDR xenograft model compared with each single agent. CONCLUSIONS: The results suggest that the TRAS + PER combination may be effective in T-DM1-resistant cancer cells where HER2 overexpression is maintained.

Acquired Resistance to Alectinib in ALK-Rearranged Lung Cancer due to ABCC11/MRP8 Overexpression in a Clinically Paired Resistance Model.

Alectinib is used as a first-line treatment for anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). Whereas other ALK inhibitors have been reported to be involved in resistance to ATP-binding cassette (ABC) transporters, no data are available regarding the association between resistance to alectinib and ABC-transporters. To investigate whether ABC-transporters contribute to alectinib resistance, ABC-transporter expression in alectinib-resistant cell lines derived from a patient with ALK-rearranged NSCLC and from H2228 lung cancer cells was evaluated and compared with that in each parent cell type. ATP-binding cassette subfamily C member 11 (ABCC11) expression was significantly increased in alectinib-resistant cell lines compared with that in alectinib-sensitive lines. ABCC11 inhibition increased sensitivity to alectinib in vitro ABCC11-overexpressing cells were established by transfection of an ABCC11 expression vector into H2228 cells, while control cells were established by transfecting H2228 cells with an empty vector. ABCC11-overexpressing cells exhibited decreased sensitivity to alectinib compared with that of control cells in vitro Moreover, the tumor growth rate following alectinib treatment was higher in ABCC11-overexpressing cells than that in control cells in vivo In addition, the intracellular alectinib concentration following exposure to 100 nmol/L alectinib was significantly lower in the ABCC11-overexpressing cell line compared with that in control cells. This is the first preclinical evidence showing that ABCC11 expression may be involved in acquired resistance to alectinib.

YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation.

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.

Molecular targeting of HER2-overexpressing biliary tract cancer cells with trastuzumab emtansine, an antibody-cytotoxic drug conjugate.

PURPOSE: Trastuzumab emtansine (T-DM1) provides clinical benefit in breast cancers overexpressing human epidermal growth factor receptor 2 (HER2). However, its efficacy against biliary tract cancers (BTC) has not been evaluated. In this study, the effectiveness of T-DM1 in various BTC cell lines and xenograft models with different levels of HER2 expression was investigated. METHODS: HER2 expression status in xenografts and patient tissue microarrays was assessed by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Cell-surface HER2 expression levels and cell growth inhibition in response to T-DM1 were examined in 17 BTC cell lines. The antitumor activity of T-DM1 was evaluated in four xenograft mouse models with different levels of HER2 expression. The effects of T-DM1 on HER2 signaling, antibody-dependent cell-mediated cytotoxicity (ADCC), cell cycle, and apoptosis were assessed in vitro. RESULTS: Cell-surface expression of HER2 was observed in both gallbladder carcinoma and cholangiocarcinoma tissues. The anti-proliferative activity of T-DM1 was higher in BTC cell lines and breast cancer cell lines with higher levels of HER2 expression. The HER2 status (IHC score|HER2-to-CEP17 ratio by FISH testing) of each BTC xenograft was 3 +|8.3 for KMCH-1, 2 +|4.7 for Mz-ChA-1, 1 +/0|1.4 for OCUG-1, and 0|1.1 for KKU-100, and T-DM1 showed antitumor activity in proportion to the HER2 status. T-DM1 inhibited HER2 signaling and induced ADCC, mitotic arrest, and apoptosis in KMCH-1 cells. CONCLUSIONS: T-DM1 exhibited preclinical activity in HER2-overexpressing BTC. Further evaluation in clinical studies is warranted.