Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser⁸⁹⁷, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.     

Bruton’s Tyrosine Kinase Targeting in Multiple Myeloma

Multiple myeloma (MM), a clonal plasma cell disorder, disrupts the bones’ hematopoiesis and microenvironment homeostasis and ability to mediate an immune response against malignant clones. Despite prominent survival improvement with newer treatment modalities since the 2000s, MM is still considered a non-curable disease. Patients experience disease recurrence episodes with clonal evolution, and with each relapse disease comes back with a more aggressive phenotype. Bruton’s Tyrosine Kinase (BTK) has been a major target for B cell clonal disorders and its role in clonal plasma cell disorders is under active investigation. BTK is a cytosolic kinase which plays a major role in the immune system and its related malignancies. The BTK pathway has been shown to provide survival for malignant clone and multiple myeloma stem cells (MMSCs). BTK also regulates the malignant clones’ interaction with the bone marrow microenvironment. Hence, BTK inhibition is a promising therapeutic strategy for MM patients. In this review, the role of BTK and its signal transduction pathways are outlined in the context of MM.     

THZ531 Induces a State of BRCAness in Multiple Myeloma Cells: Synthetic Lethality with Combination Treatment of THZ 531 with DNA Repair Inhibitors

Multiple myeloma (MM) is a hematological disease marked by abnormal growth of B cells in bone marrow. Inherent chromosomal instability and DNA damage are major hallmarks of MM, which implicates an aberrant DNA repair mechanism. Studies have implicated a role for CDK12 in the control of expression of DNA damage response genes. In this study, we examined the effect of a small molecule inhibitor of CDK12–THZ531 on MM cells. Treatment of MM cells with THZ531 led to heightened cell death accompanied by an extensive effect on gene expression changes. In particular, we observed downregulation of genes involved in DNA repair pathways. With this insight, we extended our study to identify synthetic lethal mechanisms that could be exploited for the treatment of MM cells. Combination of THZ531 with either DNA-PK inhibitor (KU-0060648) or PARP inhibitor (Olaparib) led to synergistic cell death. In addition, combination treatment of THZ531 with Olaparib significantly reduced tumor burden in animal models. Our findings suggest that using a CDK12 inhibitor in combination with other DNA repair inhibitors may establish an effective therapeutic regimen to benefit myeloma patients.     

Targeting Casein Kinase 1 (CK1) in Hematological Cancers

The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials.     

The Significance of mRNA in the Biology of Multiple Myeloma and Its Clinical Implications

Multiple myeloma (MM) is a genetically complex disease that results from a multistep transformation of normal to malignant plasma cells in the bone marrow. However, the molecular mechanisms responsible for the initiation and heterogeneous evolution of MM remain largely unknown. A fundamental step needed to understand the oncogenesis of MM and its response to therapy is the identification of driver mutations. The introduction of gene expression profiling (GEP) in MM is an important step in elucidating the molecular heterogeneity of MM and its clinical relevance. Since some mutations in myeloma occur in non-coding regions, studies based on the analysis of mRNA provide more comprehensive information on the oncogenic pathways and mechanisms relevant to MM biology. In this review, we discuss the role of gene expression profiling in understanding the biology of multiple myeloma together with the clinical manifestation of the disease, as well as its impact on treatment decisions and future directions.     

Targeting AKT in ER-Positive HER2-Negative Metastatic Breast Cancer: From Molecular Promises to Real Life Pitfalls?

The AKT protein kinase plays a central role in several interconnected molecular pathways involved in growth, apoptosis, angiogenesis, and cell metabolism. It thereby represents a therapeutic target, especially in hormone receptor-positive (HR) breast cancers, where the PI3K/AKT signaling pathway is largely hyperactivated. Moreover, resistance to therapeutic classes, including endocrine therapy, is associated with the constitutive activation of the PI3K/AKT pathway. Improved knowledge on the molecular mechanisms underlying resistance to endocrine therapy has led to the diversification of the therapeutic arsenal, notably with the development of PI3K and mTOR inhibitors, which are currently approved for the treatment of advanced HR-positive breast cancer patients. AKT itself constitutes a novel pharmacological target for which AKT inhibitors have been developed and tested in clinical trials. However, despite its pivotal role in cell survival and anti-apoptotic mechanisms, as well as in endocrine therapy resistance, few drugs have been developed and are available for clinical practice. The scope of the present review is to focus on the pivotal role of AKT in metastatic breast cancer through the analysis of its molecular features and to discuss clinical implications and remaining challenges in the treatment of HR-positive metastatic breast cancer.     

Management of Myeloma Bone Lesions

Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal plasma–cell proliferation. The survival and prognosis of this condition have been significantly improved by treatment with active anti-MM drugs such as bortezomib or lenalidomide. Further, the discovery of novel agents has recently paved the way for new areas of investigation. However, MM, including myeloma-related bone diseases, remains fatal. Bone disease or bone destruction in MM is a consequence of skeletal involvement with bone pain, spinal cord compression, and bone fracture resulting from osteolytic lesions. These consequences affect disease outcomes, including patients’ quality of life and survival. Several studies have sought to better understand MM bone disease (MBD) through the classification of its molecular mechanisms, including osteoclast activation and osteoblast inhibition. Bisphosphonates and the receptor activator of the nuclear factor-kappa B (NF-κB) ligand (RANKL) inhibitor, denosumab, prevent skeletal-related events in MM. In addition, several other bone-targeting agents, including bone-anabolic drugs, are currently used in preclinical and early clinical evaluations. This review summarizes the current knowledge of the pathogenesis of MBD and discusses novel agents that appear very promising and will soon enter clinical development.     

Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients

Unlike solid-tumor patients, a disappointingly small subset of multiple myeloma (MM) patients treated with checkpoint inhibitors derive clinical benefits, suggesting differential participation of inhibitory receptors involved in the development of T-cell-mediated immunosuppression. In fact, T cells in MM patients have recently been shown to display features of immunosenescence and exhaustion involved in immune response inhibition. Therefore, we aimed to identify the dominant inhibitory pathway in MM patients to achieve its effective control by therapeutic interventions. By flow cytometry, we examined peripheral blood (PB) CD4 T cell characteristics assigned to senescence or exhaustion, considering PD-1, CTLA-4, and BTLA checkpoint expression, as well as secretory effector function, i.e., capacity for IFN-γ and IL-17 secretion. Analyses were performed in a total of 40 active myeloma patients (newly diagnosed and treated) and 20 healthy controls. At the single-cell level, we found a loss of studied checkpoints’ expression on MM CD4 T cells (both effector (Teff) and regulatory (Treg) cells) primarily at diagnosis; the checkpoint deficit in MM relapse was not significant. Nonetheless, PD-1 was the only checkpoint distributed on an increased proportion of T cells in all MM patients irrespective of disease phase, and its expression on CD4 Teff cells correlated with adverse clinical courses. Among patients, the relative defect in secretory effector function of CD4 T cells was more pronounced at myeloma relapse (as seen in declined Th1/Treg and Th17/Treg cell rates). Although the contribution of PD-1 to MM clinical outcomes is suggestive, our study clearly indicated that the inappropriate expression of immune checkpoints (associated with dysfunctionality of CD4 T cells and disease clinical phase) might be responsible for the sub-optimal clinical response to therapeutic checkpoint inhibitors in MM.     

Novel Dual PI3K/mTOR Inhibitor,Apitolisib (GDC-0980), Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells

Deregulated PI3K/AKT/mTOR signalling commonly exists in glioblastoma, making this axis an attractive target for therapeutic manipulation. Given that activation of PI3K/AKT/mTOR promotes tumour growth, metastasis, and resistance to anticancer therapies, mTOR inhibitors show promise in the treatment of cancer. The aim of this study was to investigate the underlying mechanism of novel dual PI3K/mTOR inhibitor, Apitolisib (GDC-0980), in A-172 and U-118-MG GBM tumour cell line suppression. It has been demonstrated that GDC-0980 induces time- and dose-dependent cytotoxicity and apoptosis in investigated glioma cell lines. In our study, the strongest induction of apoptosis was exhibited in the A-172 line after 48 h of incubation with 20 µM GDC-0980, where we observed 46.47% of apoptotic cells. In conclusion, we first discovered that dual PI3K/mTOR blockade by GDC-0980 markedly suppressed survival of human GBM cells and induced apoptosis, independent of the ER stress-mediated DR5 activation. We suggest that GDC-0980, by exerting an inhibitory effect on PERK expression, may thus block its inhibitory effect on protein synthesis, leading to intensification of translation, and this may result in an increase in apoptosis. On the other hand, CHOP stimulates protein synthesis and increases apoptosis. These findings suggest that GDC-0980 may be a candidate for further evaluation as a chemotherapeutic agent for anti-GBM therapy.     

The mTOR Signalling Pathway in Human Cancer

The conserved serine/threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin, activates S6K1 and 4EBP1, which are involved in mRNA translation. It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress signals, and essential signalling pathways, such as PI3K, MAPK and AMPK, in order to control cell growth, proliferation and survival. mTORC2 is considered resistant to rapamycin and is generally insensitive to nutrients and energy signals. It activates PKC-α and AKT and regulates the actin cytoskeleton. Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression) has been reported in many types of cancers, particularly in melanoma, where alterations in major components of the mTOR pathway were reported to have significant effects on tumour progression. Therefore, mTOR is an appealing therapeutic target and mTOR inhibitors, including the rapamycin analogues deforolimus, everolimus and temsirolimus, are submitted to clinical trials for treating multiple cancers, alone or in combination with inhibitors of other pathways. Importantly, temsirolimus and everolimus were recently approved by the FDA for the treatment of renal cell carcinoma, PNET and giant cell astrocytoma. Small molecules that inhibit mTOR kinase activity and dual PI3K-mTOR inhibitors are also being developed. In this review, we aim to survey relevant research, the molecular mechanisms of signalling, including upstream activation and downstream effectors, and the role of mTOR in cancer, mainly in melanoma.     

AKT Inhibitors: The Road Ahead to Computational Modeling-Guided Discovery

AKT, is a serine/threonine protein kinase comprising three isoforms—namely: AKT1, AKT2 and AKT3, whose inhibitors have been recognized as promising therapeutic targets for various human disorders, especially cancer. In this work, we report a systematic evaluation of multi-target Quantitative Structure-Activity Relationship (mt-QSAR) models to probe AKT’ inhibitory activity, based on different feature selection algorithms and machine learning tools. The best predictive linear and non-linear mt-QSAR models were found by the genetic algorithm-based linear discriminant analysis (GA-LDA) and gradient boosting (Xgboost) techniques, respectively, using a dataset containing 5523 inhibitors of the AKT isoforms assayed under various experimental conditions. The linear model highlighted the key structural attributes responsible for higher inhibitory activity whereas the non-linear model displayed an overall accuracy higher than 90%. Both these predictive models, generated through internal and external validation methods, were then used for screening the Asinex kinase inhibitor library to identify the most potential virtual hits as pan-AKT inhibitors. The virtual hits identified were then filtered by stepwise analyses based on reverse pharmacophore-mapping based prediction. Finally, results of molecular dynamics simulations were used to estimate the theoretical binding affinity of the selected virtual hits towards the three isoforms of enzyme AKT. Our computational findings thus provide important guidelines to facilitate the discovery of novel AKT inhibitors.     

Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor–bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.     

The importance of the active site histidine for the activity of epoxide- or aziridine-based inhibitors of cysteine proteases

In the present study the importance of the active site histidine residue (His) for the activity of epoxide- or aziridine-based cysteine protease inhibitors is examined theoretically. To account for all important effects, QM/MM hybrid approaches are employed which combine quantum mechanical (QM) methods that are necessary to describe bond-breaking and formation processes, with molecular mechanics (MM) methods that incorporate the influence of the protein environment. Using various model systems, the computations exclude a direct proton shift from the active site His residue to the inhibitor, but show that one water molecule is sufficient to establish a very efficient relay system. This relay system allows an easy proton transfer from the active site His residue to the inhibitor and is thus essential for the activity of both types of inhibitors. Differences between the epoxides and the aziridines are discussed, along with some implications for the rational design of optimized inhibitors. The work presented herein represents the first QM/MM study into the mode of action of these important inhibitor classes.     

Multiple Mechanisms Mediate Resistance to Sorafenib in Urothelial Cancer

Genetic and epigenetic changes in the mitogen activated protein kinase (MAPK) signaling render urothelial cancer a potential target for tyrosine kinase inhibitor (TKI) treatment. However, clinical trials of several TKIs failed to prove efficacy. In this context, we investigated changes in MAPK signaling activity, downstream apoptotic regulators and changes in cell cycle distribution in different urothelial cancer cell lines (UCCs) upon treatment with the multikinase inhibitor sorafenib. None of the classical sorafenib targets (vascular endothelial growth factor receptor 1/-receptor 2, VEGFR1/-R2; platelet-derived growth factor receptor α/-receptor β, PDGFR-α/-β; c-KIT) was expressed at significant levels leaving RAF proteins as its likely molecular target. Low sorafenib concentrations paradoxically increased cell viability, whereas higher concentrations induced G1 arrest and eventually apoptosis. MAPK signaling remained partly active after sorafenib treatment, especially in T24 cells with an oncogenic HRAS mutation. AKT phosphorylation was increased, suggesting compensatory activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Sorafenib regularly down regulated the anti-apoptotic myeloid cell leukemia 1 (Mcl-1) protein, but combinatorial treatment with ABT-737 targeting other B-cell lymphoma 2 (Bcl-2) family proteins did not result in synergistic effects. In summary, efficacy of sorafenib in urothelial cancer cell lines appears hampered by limited effects on MAPK signaling, crosstalk with further cancer pathways and an anti-apoptotic state of UCCs. These observations may account for the lack of efficacy of sorafenib in clinical trials and should be considered more broadly in the development of signaling pathway inhibitors for drug therapy in urothelial carcinoma.     

Synergistic Effects of Venetoclax and Daratumumab on Antibody-Dependent Cell-Mediated Natural Killer Cytotoxicity in Multiple Myeloma

The prognosis of multiple myeloma (MM) has drastically improved owing to the development of new drugs, such as proteasome inhibitors and immunomodulatory drugs. Nevertheless, MM is an extremely challenging disease, and many patients are still refractory to the existing therapies, thus requiring new treatment alternatives. Venetoclax is a selective, orally bioavailable inhibitor of BCL-2 that shows efficacy in MM not only as a single agent but also in combination therapy, especially for MM patients with translocation t(11;14). However, many patients are refractory to this drug. Here, we treated the MM cell lines KMS12PE and KMS27 with a combination treatment of venetoclax targeting BCL-2 and daratumumab targeting CD38 to evaluate the synergistic cytotoxicity of these drugs in vitro. MM cell lines were co-cultured with natural killer (NK) cells at an effector:target ratio of 0.3:1 in the presence of serial concentrations of daratumumab and venetoclax, and the resulting apoptotic MM cells were detected by flow cytometry using annexin V. These results indicated that the antibody-dependent cell-mediated NK cytotoxicity was enhanced in KMS12PE and KMS27 cells harboring t(11;14) with a high BCL-2 expression, suggesting that the combination treatment of venetoclax and daratumumab should be especially effective in patients with these characteristics.     

Development of Novel Immunotherapies for Multiple Myeloma

Multiple myeloma (MM) is a disorder of terminally differentiated plasma cells characterized by clonal expansion in the bone marrow (BM). It is the second-most common hematologic malignancy. Despite significant advances in therapeutic strategies, MM remains a predominantly incurable disease emphasizing the need for the development of new treatment regimens. Immunotherapy is a promising treatment modality to circumvent challenges in the management of MM. Many novel immunotherapy strategies, such as adoptive cell therapy and monoclonal antibodies, are currently under investigation in clinical trials, with some already demonstrating a positive impact on patient survival. In this review, we will summarize the current standards of care and discuss major new approaches in immunotherapy for MM.     

BH3 Mimetics in Hematologic Malignancies

Hematologic malignancies (HM) comprise diverse cancers of lymphoid and myeloid origin, including lymphomas (approx. 40%), chronic lymphocytic leukemia (CLL, approx. 15%), multiple myeloma (MM, approx. 15%), acute myeloid leukemia (AML, approx. 10%), and many other diseases. Despite considerable improvement in treatment options and survival parameters in the new millennium, many patients with HM still develop chemotherapy-refractory diseases and require re-treatment. Because frontline therapies for the majority of HM (except for CLL) are still largely based on classical cytostatics, the relapses are often associated with defects in DNA damage response (DDR) pathways and anti-apoptotic blocks exemplified, respectively, by mutations or deletion of the TP53 tumor suppressor, and overexpression of anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family. BCL2 homology 3 (BH3) mimetics represent a novel class of pro-apoptotic anti-cancer agents with a unique mode of action—direct targeting of mitochondria independently of TP53 gene aberrations. Consequently, BH3 mimetics can effectively eliminate even non-dividing malignant cells with adverse molecular cytogenetic alterations. Venetoclax, the nanomolar inhibitor of BCL2 anti-apoptotic protein has been approved for the therapy of CLL and AML. Numerous venetoclax-based combinatorial treatment regimens, next-generation BCL2 inhibitors, and myeloid cell leukemia 1 (MCL1) protein inhibitors, which are another class of BH3 mimetics with promising preclinical results, are currently being tested in several clinical trials in patients with diverse HM. These pivotal trials will soon answer critical questions and concerns about these innovative agents regarding not only their anti-tumor efficacy but also potential side effects, recommended dosages, and the optimal length of therapy as well as identification of reliable biomarkers of sensitivity or resistance. Effective harnessing of the full therapeutic potential of BH3 mimetics is a critical mission as it may directly translate into better management of the aggressive forms of HM and could lead to significantly improved survival parameters and quality of life in patients with urgent medical needs.     

Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.