Search

Supplementary Figures and Legends

Supplementary Video 2

Supplementary Tables

Supplementary Video 5

Supplementary Video 4

Supplementary Video 1

Supplementary Video 6

Supplementary Video 3

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF–MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy–resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors.Significance:This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601

Cyclin-dependent kinases (CDKs) 4 and 6 inhibitors (CDK4/6is) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function and are exquisitely sensitive to CDK4/6is. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6is and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90–CDC37 complex. In contrast, CDK4/6is and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6is and CDK4/6i-derived degraders and the need for new inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics. Poulikakos and colleagues demonstrate that thermostable CDK6 complexes promote resistance to therapeutic targeting of CDK4/6 in cancer.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF–MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy–resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. Significance: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors. See related commentary by Zhang and Bollag, p. 1620. This article is highlighted in the In This Issue feature, p. 1601

Label-free miniaturized optical sensors can have a tremendous impact on highly sensitive and scalable point-of-care (PoC) diagnostics by monitoring in real-time molecular interactions without any labels. However, current biophotonic platforms are limited by complex optical and external readout equipment, precluding their use in a PoC setting. In this work, we address this challenge by using a first of its kind fully integrated biosensing electronic–photonic system-on-chip (EPSoC) utilizing microring resonators (MRRs) co-integrated with on-chip electronics in a high volume advanced electronic process. We demonstrate the sensing capabilities of the EPSoC, implemented in GF 45-nm RFSOI, by monitoring real-time binding events of streptavidin molecules, unlocking the door toward true monolithic lab-on-chip (LoC) photonic sensors for PoC applications.

In the present study we investigated the expression and the functional role of mechanosensitive polycystins in renal cell carcinoma (RCC). In 115 RCC patients we evaluated the protein expression of polycystin-1 (PC1), polycystin-2 (PC2), VEGF and protein components of the PI3K/Akt/mTOR pathway, which have been implicated both in RCC and polycystic kidney disease. PC1 and PC2 demonstrated reduced expression throughout the RCC tissue compared to the adjacent normal tissue. PC1 and PC2 revealed high expression when they were associated with higher grade and decreased 5-year survival respectively. PC1 and PC2 were positively correlated with p110γ subunit of PI3K and high PC1 expressing cells tended to display activation/phosphorylation of Akt. There was also a positive association between PC1 and VEGF expression, whereas PC1 augmented the tumor's microvascular network in stage IV carcinomas. In human RCC cells, functional inhibition of PC1 resulted in upregulation of the PI3K/Akt/mTOR pathway, enhanced cell proliferation and led to inhibition of cell migration. Conclusively, aberrant PC1 regulation is associated with increased angiogenesis and features of advanced disease in RCC tissues.

Activating RAS mutations constitute the majority of recurrent mutations found in MAPK-driven human tumors. However, RAS mutants have long been considered undruggable, and therefore efforts have been predominantly focused on targeting downstream MAPK signaling. We and others have shown that, MAPK inhibition using MEK inhibitors (MEKi) have limited effectiveness in such tumors due to adaptive resistance. A major mechanism of adaptive resistance involves relief of negative feedback that promotes reactivation of RTK signaling, which in turn activates RAS and results in MAPK rebound. The recent development of direct (i.e. RAS(G12C)) and indirect (i.e. SHP2i, SOSi) inhibitors of RAS have uncovered the dependence of activity of RAS(G12C) and of other RAS mutants on upstream signaling. We and others have previously characterized sensitivity of various RAS mutants in cancer to either SHP2 inhibition (SHP2i), or to combination of SHP2i with MEKi, but a systematic characterization of most RAS mutants commonly expressed in cancer has been lacking. In this study, by combining biochemical and chemical genetic approaches we identified two classes of RAS-mutants, based on their degree of dependency on RTK/SHP2 signaling and adaptation to MEK inhibition. One class of RAS mutants, including most RASQ61X, show complete independence from RTK/SHP2 signaling and are fully resistant to either SHP2i alone or in combination with MEKi. The other class includes the majority of RAS mutants and is characterized by relative resistance to SHP2i, or the combination of SHP2i plus MEKi, compared to cells expressing wild-type RAS, including normal cells. Early clinical data using single agent SHP2i or combinations of SHP2i with MEKi for the treatment of patients with RAS-mutant tumors suggested modest activity as well as dose-limiting on-target (i.e. MAPK pathway related) toxicities. Our findings predict that achieving an acceptable therapeutic index by either approach for treatment of RAS-mutant tumors will be challenging, and they support instead SHP2i-based therapeutic efforts in patients with MAPK-driven tumors expressing wild-type RAS, or in combination with RAS-mutant selective inhibitors. Collectively this work provides a framework for the design of SHP2i-based treatments for patients with MAPK-driven tumors, the majority of which currently have no effective targeted therapeutic options. Citation Format: Beau Baars, Christos Adamopoulos, Ana Orive-Ramos, Stuart A. Aaronson, Poulikos I. Poulikakos. RTK/SHP2 dependency and feedback adaptation to MEK inhibition defines sensitivity of RAS-mutant proteins to SHP2 inhibitor-based therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3413.

Oncogenic alterations of the RAS/RAF/MEK/ERK signaling pathway drives growth of over 40% of human tumors, frequently due to activating mutations of components of the pathway, such as RAS or BRAF. Targeting the pathway using BRAF inhibitors (BRAFi) in combination with MEK inhibitors (MEKi) is now the standard of care for metastatic melanoma patients harboring the BRAF(V600E) mutation. However, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including those with RAS activating mutations. The effectiveness of current clinical BRAFi relies on the fact that they bind selectively and inhibit the mutationally activated monomeric form of the BRAF(V600E) kinase in tumors but not wild-type BRAF in normal tissue where BRAF signals in its dimeric form. Yet, the development of adaptive resistance, frequently due to RAF dimerization, limits BRAFi therapeutic effectiveness. Another class of BRAFi equipotent for both monomeric and dimeric BRAF have been developed, but are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues as well. Recently, we identified and characterized a novel class of BRAFi that preferentially binds and inhibits dimeric BRAF over monomeric BRAF(V600E) (i.e. RAF dimer-selective inhibitors). Furthermore, we rationally designed a combinatorial approach utilizing a BRAF monomer-selective plus a BRAF dimer-selective inhibitor plus a MEK inhibitor, that potently disrupts the BRAF/MEK complex, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index. This triple combination potently suppressed tumor growth of multiple BRAF(V600E) colorectal cancer and melanoma models resistant to the current clinical BRAFi and MEKi combinations both in vitro and in vivo. To design effective therapeutic approaches for RAS-mutated tumors, we also assessed the effectiveness of RAF and MEK inhibitors belonging to diverse biochemical and structural classes, either alone or in combination in RAS-mutant tumor models and normal cells with wild-type RAS signaling. We found that RAF dimer-selective inhibitors potently antagonized MAPK signaling and growth in various RAS mutant cancer models as compared to normal cells. We found further that RAF and MEK inhibitor combinations belonging to certain biochemical classes resulted in more potent MAPK suppression in RAS-mutant tumor as compared to wild-type RAS normal cells (“therapeutic synergy”) and should be prioritized for clinical testing. In contrast, those combinations which are more potent in normal over tumor cells (“adverse synergy”) are unlikely to be clinically successful. Thus, rationally designed combinations of selected RAFi and MEKi, based on their distinct biochemical properties, can uncover new therapeutic opportunities tailored for more effective targeting of BRAF or RAS mutant tumors. Citation Format: Ana Orive-Ramos, Christos Adamopoulos, Beau Baars, Jason Lam, Ankita Punetha, Jason Huang, Vasileios I. Petrou, Stuart A. Aaronson, Poulikos I. Poulikakos. Designing RAF and MEK inhibitor combinations based on their biochemical properties to effectively target MAPK-driven cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5734.

OBJECTIVES Periodontitis is a highly prevalent chronic inflammatory disease caused by periodontopathogens, such as Filifactor alocis. This study sought to examine the matrix metalloproteinase (MMP)-1 synthesis by monocytic and fibroblastic cells in response to F. alocis and to unravel the underlying cellular mechanisms. MATERIAL AND METHODS Gingival biopsies from periodontally healthy and periodontitis individuals were analyzed for the presence of F. alocis and MMP-1 by RT-PCR. Human gingival fibroblastic (HGF-1) and monocytic (THP-1) cells were stimulated with F. alocis in the presence and absence of a blocking toll-like receptor (TLR)2 antibody or specific inhibitors against MAPKs. MMP-1 expression and protein levels were studied by RT-PCR and ELISA, respectively. RESULTS F. alocis was highly prevalent in biopsies from periodontitis patients but barely present in the healthy gingiva. Significantly higher MMP-1 expression levels were found in the inflamed gingiva as compared with healthy biopsies. F. alocis caused a significant and dose-dependent MMP-1 upregulation in both cells. The stimulatory effect of F. alocis on MMP-1 was TLR2- and MAPK-dependent and more pronounced on THP-1 cells as compared with HGF-1 cells. CONCLUSIONS Our results demonstrate that F. alocis and MMP-1 are more prevalent at periodontitis sites. Additionally, our study provides original evidence that F. alocis can stimulate MMP-1 production by fibroblastic and monocytic cells, suggesting that F. alocis may contribute to periodontal breakdown through MMP-1. CLINICAL RELEVANCE F. alocis and MMP-1 are linked to each other and key players in periodontitis, which may have significant implications for future diagnostic and treatment strategies.

This paper presents the first fully integrated 2-D array of electronic-photonic ultrasound sensors targeting low-power miniaturized ultrasound probes for endoscopic applications. Fabricated in a zero-change 45nm CMOS-SOI technology this Electronic-Photonic System on Chip (EPSoC), utilizes micro-ring resonators (MRRs) as ultrasound sensors instead of the traditional piezoelectric or capacitive micromachined transducers (PMUTs or CMUTs). The photonic nature of the sensor enables remoting the power-hungry receive electronics outside the probe tip, lowering the power dissipation inside the human body, eliminates the electrical cabling and reduces fiber count by 4x using wavelength division multiplexed (WDM) MRR sensors coupled onto the same waveguide. The photonic sensing element demonstrates >30 MHz bandwidth, and 7.3 mV/kPa sensitivity, while consuming 0.43 mW of power.

Immediate and widely accessible diagnostics are the key for halting rapidly spreading, and potentially fatal pathogens. A low cost, scalable, real-time biosensing platform unlocks the path towards miniaturized and cost-effective Point of Care (POC) sensors. Here we solve this problem by developing the world’s first fully integrated electronic-photonic, real-time, label-free Refractive Index (RI) sensor, utilizing on-chip electronics and micro-ring resonators (MRRs) in a commercial CMOS 45nm RFSOI platform. RI sensing and real time molecular kinetics are demonstrated for the first time in a monolithic electronic-photonic system-on-chip without requiring bulky optics and off-chip electronic equipment, enabling self-contained real-time POC sensors. The sensitivity performance corresponds to a limit of detection (LoD) of a single 100nm nanoparticle, similar to the size and RI of the SARS-CoV-2 virus.

The first optical ultrasound sensor with co-integrated read-out circuitry is presented. Based on a micro-ring resonator (MRR), it has a measured 7.3mV/kPa sensitivity, 480Pa minimum detectable pressure, operating at 80% fractional bandwidth around 5MHz.

We demonstrate the world's first fully integrated electronic-photonic label-free molecular sensor in a CMOS 45RFSOI commercial process. Real-time kinetics with 100nm lipid nanoparticles, mimicking SARS-CoV-2 particles, are presented utilizing micro-ring resonators integrated with on-chip electronics.

BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical BRAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF (i.e. “BRAF monomer-selective”) providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric BRAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. We identified and characterized a novel class of BRAF inhibitors that preferentially bind and inhibit dimeric over monomeric BRAF(V600E) (i.e. “BRAF dimer-selective inhibitors”). Biochemical analysis using BRAF dimer-selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of BRAF monomer- plus BRAF dimer selective inhibitor plus a MEK inhibitor that disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical BRAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the BRAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher Therapeutic Index. Finally, off-label use of the triple combination in patients with advanced BRAF(V600E) cancers that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective BRAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Overall, our work supports the notion that for most tumors a “3-drug” combinatorial strategy will potentially be most effective: one drug targeting MAPK signaling directly, such as a MEK inhibitor (“pathway” inhibitor), one drug targeting mutated BRAF or RAS selectively in the tumor (“therapeutic index” inhibitor, e.g. BRAF monomer-selective inhibitor, RAS(G12C) inhibitors, etc) and one drug targeting components of the feedback loop that is responsible for adaptive resistance (“feedback” inhibitor, e.g. BRAF dimer-selective inhibitors, SHP2 or SOS inhibitors, etc). Our data contribute to the development of a roadmap for the treatment of MAPK-driven tumors using drug combinations tailored to the specific driver oncoprotein. Citation Format: Christos Adamopoulos, Tamer Ahmed, Peter M. Ung, Min Xiao, Stuart Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Poulikos I. Poulikakos. BRAF dimer-selective inhibitors synergize with BRAF monomer-selective inhibitors to overcome adaptive resistance and retain therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB083.

Psoriatic plaques tend to localize to the knees and elbows, areas that are particularly subject to mechanical stress resulting from bending and friction. Moreover, plaques often develop at sites of mechanical trauma or injury (Koebner phenomenon). Nevertheless, mechanotransduction has never been linked to psoriasis. Polycystins (polycystin-1, PC1; polycystin-2, PC2) are mechanosensitive molecules that function as key regulators of cellular mechanosensitivity and mechanotransduction. The aim of this in vitro study was to investigate the role of polycystins in the development of psoriasis. We showed that PC1 knockdown in HaCaT cells led to an elevated mRNA expression of psoriasis-related biomarkers Ki-67, IL-6, TNF-α, VEGF and Bcl-2, while PC1 functional inhibition was accompanied by increased cell proliferation and migration of HaCaT cells. In addition, PC1 knockdown via siRNA in HaCaT cells was followed by activation of critical molecules of the mTOR and MAPK pathways and this mTOR pathway activation was ERK-dependent. Furthermore, loss of PC1 protein expression and elevated levels of activated mTOR substrates were also observed in human samples of psoriatic plaques. Overall, our study suggests that the PC1/ERK/mTOR signaling axis represents a novel potential mechanism in psoriasis pathogenesis.

Background: Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite numerous targeted therapy options, failing anti-BCMA CAR-T cell therapy represents a challenging treatment course with dismal prognosis. 53% of relapsed/refractory (rr) MM patients have shown emerging clones harboring mutations within the MAPK signaling pathway including the targetable BRAF V600E in 7% of cases. Allosteric kinase inhibition of MAPK pathway with BRAF monomer selective inhibitors +/- MEK inhibitor have been frequently attempted with rrMM patients with equivocal outcome on both disease progression and survival. This could be ascribed to the feedback activation of the pathway via BRAF dimers induction. In solid tumor contexts, a recent study has revealed that the multi-kinase inhibitor regorafenib is a potent and selective inhibitor of dimeric BRAF. Moreover, the study demonstrated overcoming of this negative feedback by combining two inhibitors targeting BRAF in its monomeric and its dimeric form, respectively, with MEK inhibition leading to more efficacious and tolerable treatment (Adamopoulos C. et al. Cancer Discov. 2021). Here, we show the applicability, effectiveness, and tolerability of the aforementioned triple therapy strategy in hematological malignancy context to salvage post BCMA CAR-T cell therapy in a BRAF V600E rrMM patient. Material and Methods: CD138 + selected cells were collected from bone marrow (BM) and patient's plasmacytoma before and after CAR-T cell therapy relapse, respectively. Samples were processed using the standard GATK 4 workflows and a consensus mutation calling approach was used to call somatic mutations using a matched normal obtained from peripheral blood. PhyloWGS (Deshwar et al, Genome Biol 2015) was then used to perform intra-tumor heterogeneity modeling and phylogeny estimation. CD138 + selected cells from BM of rrMM patient were treated with encorafenib (50nM) and binimetinib (250 nM) +/- regorafinib (1mM) for 48 hours, cells were harvested, lysed and immunoblotted with BRAF V600E and pERK antibodies. In parallel, rrMM patient BM selected CD138 + cells' sensitivity to MAPK inhibitors were assessed by Travera. Cells are incubated with different inhibitors at varying concentrations and combinations for 15 hours before having their single-cell mass distributions measured. Sensitivity is then characterized by the relative statistical difference (Hellinger distance) between conditions and untreated controls. A rrMM patient was started on targeted triple therapy using the following combination: BRAF monomer-inhibitor, dabrafenib (100mg, orally twice daily), a MEK-inhibitor, trametinib (1.5mg, orally for 21/28 days daily), and a BRAF dimer inhibitor, regorafenib (40mg, orally once daily). Results: Whole Exome Sequencing (WES) showed a BRAF V600E mutation was present prior to CAR-T cell therapy and persisted through subsequent treatments. The variant allele frequency of the mutation was 41% post CAR-T cell therapy (Figure 1A). Western blot of CD138 + cells confirmed BRAF V600E mutation with a specific antibody, and showed ERK phosphorylation, which was inhibited with trametinib. Longer exposure to trametinib confirmed a feedback loop activating MAPK which was abrogated with the BRAF dimer inhibitor regorafenib (Figure 1B). Travera analysis showed CD138 + cells' sensitivity to the combination of trametinib at 5mM, and dabrafenib at 1µM. Adding regorafenib at 1µM could slightly improve the response compared to the double combination. (Figure 1C). Clinically, three months of the triple therapy administration has led to prompt reduction in subcutaneous skin lesions as well as 80% reduction in free lambda chain (Figure 1D). Furthermore, the patient exhibited good tolerance to all three medications with minimal side effects (grade 1 fatigue). The treatment regimen allowed the patient to carry out activities of daily living and resume work. Conclusion: We have shown the applicability, effectiveness, and tolerability of the triple MAPK inhibition in a hematological malignancy context to salvage post BCMA CAR-T cell therapy in a rrMM patient with BRAF V600E mutation. Also, we have revealed the power of routine WES in tracking clonal evolution, and identifying targetable mutations which allows tailoring of therapeutics for MM patients. Figure 1 Figure 1. Disclosures Stevens: Travera: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Glycative stress from endogenous and exogenous advanced glycation end-products (AGEs) has been implicated to cancer development and progression. Dicarbonyl compounds, the main AGE precursors and crosslinked AGE forms may directly react with proteins, lipids and nucleic acids, modify their structure and affect tissue microenvironment. They may also induce elevation of reactive oxygen species (ROS) and enhance cellular oxidative stress, an important regulator of cancer hallmarks. Moreover, the activation of AGE-receptor for AGE (RAGE) signalling pathways mediates inflammation, oxidative stress, autophagy and apoptosis leading to genomic instability and cancer initiation. Here, we provide evidence on the impact of glycative stress in promoting human tumorigenesis and we discuss the potential application of anti-glycating agents, RAGE and glyoxalase-1 inhibitors in cancer prevention.

BRAF kinase, a critical component of the RAS/MAPK signaling pathway, requires dimerization for its catalytic activity, whereas the oncoprotein BRAF(V600E) can be catalytically active as a monomer. Current clinical RAF inhibitors selectively bind and inhibit monomeric over dimeric BRAF providing a high therapeutic index and are now standard practice in combination with MEK inhibitors in the treatment of patients harboring BRAF(V600E) tumors. However adaptive resistance due to RAF dimerization limits their effectiveness. Recently, RAF inhibitors that equipotently inhibit both monomeric and dimeric RAF have been developed, but they are predicted to have lower therapeutic index due to inhibition of dimeric wild-type BRAF in normal tissues. Here we identify and characterize a third class of RAF inhibitors that preferentially binds and inhibits dimeric over monomeric BRAF(V600E). Using long-timescale molecular dynamics (MD) simulations, we observed distinct dynamic features of RAF inhibitors bound to monomeric and dimeric BRAF, thus providing a structural explanation for the selectivity of these drugs. Biochemical analysis using RAF dimer selective inhibitors revealed that the two forms of dimeric and monomeric BRAF(V600E) differ in their conformation in their active site and their strength of interaction with MEK. Further, to maximally inhibit BRAF(V600E) signaling in tumors while retaining a broad therapeutic index, we assessed the triple combination of RAF monomer- plus RAF dimer selective inhibitor plus a MEK inhibitor that potently disrupts the BRAF-MEK complex. The triple combination potently suppressed tumor growth in multiple colorectal and melanoma BRAF(V600E) cell line- and in vivo models that were resistant to the current clinical RAF and MEK inhibitor combination. Strikingly, while the double combination treatment with the RAF dimer-selective inhibitor and MEK inhibitor was associated with a significant gradual weight loss in vivo, the triple combination showed no weight loss or other apparent toxicities, indicating a higher therapeutic index. Finally, off-label use of the triple combination in a patient with a stage IV BRAF(V600E) colorectal cancer that progressed on standard therapies achieved durable tumor control with minimal toxicities. Thus, a rationally designed combinatorial approach of conformation-selective RAF and MEK inhibitors may be a highly effective and well tolerated therapeutic strategy for patients with BRAF(V600E) tumors. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Maxwell R. Tucker, Peter M. Ung, Min Xiao, Zoi Karoulia, Xuewei Wu, Stuart A. Aaronson, Celina Ang, Vito Rebecca, Avner Schlessinger, Meenhard Herlyn, Qi Wang, David E. Shaw, Poulikos I. Poulikakos. A strategy for effective targeting of oncogenic BRAF signaling with increased therapeutic index using conformation-selective RAF and MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 78.

CDK4 and CDK6 kinases are master regulators of cell cycle progression, and they have thus been attractive targets for cancer therapy. CDK4/6 inhibitors (CDK4/6i) showed significant clinical effectiveness in ER+ breast cancers, and three CDK4/6i are now FDA-approved for this indication. However, in several tumor types, CDK4/6i have only been modestly effective, but the underlying mechanism(s) accounting for the discrepancy have remained elusive. To identify the basis of resistance to CDK4/6i, we employed a variety of biochemical, genetic and proteomic approaches, we developed a potent and selective CDK4/6-directed degrader (PROTAC) to investigate in-cell binding of compounds and validated our findings in large tumor and clinical-based data. Our studies revealed that tumor response to CDK4/6i is critically determined by the expression of CDK6. Tumors with low CDK6 expression (CDK6-low) depend on CDK4, and are uniformly sensitive to CDK4/6i. CDK6-low tumors include both entire cancer types (e.g. Ewing Sarcomas, MCL), as well as subgroups of large tumor types, such as portion of non-small cell lung adenocarcinomas (NSCLC). We further validated this finding in NSCLC patients previously treated with CDK4/6i in a clinical trial. In contrast, tumors that express both CDK4 and CDK6 universally depend on CDK6, which is expressed as either CDK4/6i-sensitive CDK6 (CDK6-S) or CDK4/6-resistant CDK6 (CDK6-R) in different cells. Using a CDK4/6-directed PROTAC, we further found that CDK4/6i binds strongly CDK6-S but weakly CDK6-R, indicating different conformations adopted by CDK6 in the two states. An unbiased proteomic analysis identified binding of components of the HSP90/CDC37 complex associated with the state of CDK6: in tumors in which CDK6 is expressed as a thermo-unstable, strong HSP90/CDC37-client, CDK4/6i (and consequently CDK4/6 PROTACs) bind and potently inhibit CDK6 and downstream Rb/E2F signaling. In contrast, tumors resistant to CDK4/6i express CDK6 as a thermostable, weak HSP90/CDC37-client that binds weakly to CDK4/6i. Our data uncover the expression state of CDK6 and its dependence on the HSP90/CDC37 complex as a critical determinant of tumor response to CDK4/6i. We further identify low CDK6 expression as a molecular predictor of tumor sensitivity to current CDK4/6i, indicating a potential biomarker for selection of patients that are more likely to benefit from these drugs. Finally, our findings underline the need for novel inhibitors targeting the thermostable CDK6-R to be used for the treatment of the large number of patients with of Rb-proficient solid tumors that are resistant to current clinical CDK4/6i. Thus, this unexpected dualism in the dependence of CDK6 kinase on HSP90 explains resistance of a large portion of tumors to current CDK4/6i, and provides a roadmap for developing more effective CDK4/6-directed pharmacologic strategies for cancer therapy. Citation Format: Xuewei Wu, Xiaobao Yang, Yan Xiong, Ruitong Li, Takahiro Ito, Tamer Ahmed, Zoi Karoulia, Christos Adamopoulos, Hong Wang, Li Wang, Ling Xie, Beatrix Ueberheide, Stuart Aaronson, Xian Chen, William Sellers, Sean Buchanan, Jian Jin, Poulikos I. Poulikakos. Tumor resistance to CDK4/6 inhibitors and degraders determined by the expression state of CDK6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 41.

Human gliomas represent the most common type of intracranial tumors, with highest morbidity and mortality. They are characterized by excessive invasiveness and cell proliferation while their unclear boundaries predispose to tumor recurrence soon after conventional treatment. Elucidation of the molecular mechanisms implicated in their development and/or treatment resistance is highly demanded. The high-mobility group box 1 (HMGB1) protein, a highly conserved nuclear protein that functions as a chromatin-binding factor, facilitating nucleosome stabilization and regulating gene transcription, has been implicated in glioma formation and progression. Extracellular released HMGB1 binds to high-affinity receptors, including the receptor for advanced glycation end-products (RAGE) and toll-like receptor (TLR)-2, TLR-4, and TLR-9. Upon receptor binding, HMGB1 triggers the activation of key signaling pathways and immune responses, involved in the regulation of cell growth, differentiation, motility, and apoptosis. Based on the type of receptor and/or cell, HMGB1 is capable to promote oncogenesis or suppress tumor growth, thus affecting treatment efficacy. Herein, we discuss recent evidence implicating HMGB1 in glioma cell differentiation, proliferation, and metastasis with both clinical and prognostic significance. In addition, potential therapeutic approaches to target this protein in order to reduce chemoresistance of glioma cells are also addressed.

Emerging evidence indicates that accumulation of advanced glycation end products (AGEs) in human tissues may contribute to cell injury, inflammation and apoptosis through induction of endoplasmic reticulum (ER) stress. Human metabolism relies on ER homeostasis for the coordinated response of all metabolic organs by controlling the synthesis and catabolism of various nutrients. In vitro studies have demonstrated AGE-induced enhancement of unfolded protein response (UPR) in different cell types including endothelial, neuronal, pancreatic cells and podocytes, suggesting this crosstalk as an underlying pathological mechanism that contributes to metabolic diseases. In this minireview, we describe in vivo studies undertaken by our group and others that demonstrate the diverse systemic effects of AGEs in ER stress induction in major metabolic tissues such as brain, kidney, liver and pancreas of normal mice. Administration of high-AGEs content diet to normal mice for the period of 4 weeks upergulates the mRNA and protein levels of ER chaperone Bip (GRP78) indicative of UPR initiation in all major metabolic organs and induces activation of the pivotal transcription factor XBP1 that regulates glucose and lipid metabolism. Furthermore, animals with genetic ablation of UPR-activated transcription factor C/EBP homologous protein CHOP allocated in high-AGEs diet, exhibited relative resistance to UPR induction (BiP levels) and XBP1 activation in major metabolic organs. Since CHOP presents a critical mediator that links accumulation and aggregation of unfolded proteins with induction of oxidative stress and ER stress-related apoptosis, it is revealed as an important molecular target for the management of metabolic diseases.

Normal extension and skull expansion is a synchronized process that prevails along the osteogenic intersections of the cranial sutures. Cranial sutures operate as bone growth sites allowing swift bone generation at the edges of the bone fronts while they remain patent. Premature fusion of one or more cranial sutures can trigger craniosynostosis, a birth defect characterized by dramatic manifestations in appearance and functional impairment. Up until today, surgical correction is the only restorative measure for craniosynostosis associated with considerable mortality. Clinical studies have identified several genes implicated in the pathogenesis of craniosynostosis syndromes with useful insights into the underlying molecular signaling events that determine suture fate. In this review, we exploit the intracellular signal transduction pathways implicated in suture pathobiology, in an attempt to identify key signaling molecules for therapeutic targeting., Highlights • Cranial sutures operate as bone growth sites. • Premature fusion of one or more cranial sutures can trigger craniosynostosis. • Several genes are involved in the pathogenesis of craniosynostosis syndromes. • An array of molecular signaling events determine suture fate. • Herein, the signal transduction pathways implicated in suture pathobiology are discussed.

Background Neuroinflammation, impaired brain insulin signaling, and neuronal apoptosis may be interrelated in the pathophysiology of people with Alzheimer disease (AD) and diabetes, either type 1 or 2 diabetes (T1D or T2D, respectively). Methods We studied 116 patients: 41 with AD, 20 with T1D, 21 with T2D, and 34 healthy controls. The number (n) of cytokine-secreting peripheral blood mononuclear cells (PBMCs) before and after mitogenic stimulation was determined for interleukin 1β (IL1β), interleukin 6 (IL6), tumor necrosis factor (TNF) by the enzyme-linked-immuno-spot assay. Serum concentrations of C-reactive protein (CRP) and Fas ligand (FASLG) were determined by ELISA. Results The studied subgroups did not differ in sex but differed in age. Higher CRP concentrations were detected in the AD group than in the T1D group (P = 0.02) and lower in controls (P < 0.001). The nPBMCs was higher in AD patients after stimulation than in basal conditions: after stimulation in nTNF (P < 0.001 vs T2D; P < 0.001 vs T1D; P = 0.001 vs control), nIL6 (P = 0.039 vs T2D; P < 0.001 vs T1D; P = 0.007 vs control), and nIL1β (P = 0.03 vs control). The nPBMCs increased after stimulation with ΡΜA in all the subgroups (P < 0.001). FASLG in the AD group displayed statistically higher concentrations than in all other subgroups (P < 0.001 vs T2D; P < 0.001 vs T1D; P = 0.012 vs control). The nPBMCs was positively correlated with plasma concentrations of FASLG in the AD subgroup. Conclusions Patients with AD display a low-grade systemic inflammation compared to people with diabetes. The FAS–FASLG pathway has a potential role because FASLG concentrations are positively correlated with the inflammatory response in AD. However, this positive correlation cannot be seen in people with diabetes, at least not with the apoptotic markers used in the present study.

The actions of estrogens are mediated by estrogen receptors, ERα and ERβ. Recent genomic landscaping of ERα- and ERβ-binding sites has revealed important distinctions regarding their transcriptional activity. ERβ and its isoforms have been correlated with endocrine treatment responsiveness in breast tumors, while post-translational modifications, receptor dimerization patterns, and subcellular localization are increasingly recognized as crucial modulators in prostate carcinogenesis. Androgen receptor (AR) is essential for the development and progression of prostate cancer as well as of certain breast cancer types. The balance between the activity of these two hormone receptors and their molecular interactions in different clinical settings is influenced by several coregulators. This comprises a dynamic regulatory network enhancing or limiting the activity of AR-directed treatments in breast and prostate tumorigenesis. In this review, we discuss the molecular background regarding the therapeutic targeting of androgen/estrogen receptor crosstalk in breast and prostate cancer.

Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties and cell proliferation.

Membrane receptors constitute novel targets during current treatment of metastatic colorectal cancer (CRC) due to the fact that their aberrant expression/activity favors cancer cell properties. Protein trafficking is responsible for the correct targeting of membrane receptors to the apical and basolateral surfaces, as well as to the adherent and tight junctions of the cell. Impaired availability or distribution of these receptors along the plasma membrane is not only associated with defective cellular homeostasis and tumor progression, but also to emerging mechanisms of resistance to CRC-targeted therapy. The present review describes how protein trafficking facilitates invasion and metastasis of CRC cells and focuses on receptor tyrosine kinases (RTKs) endocytic transport, providing thoughts for surpassing RTKs-centered mechanisms of resistance.

Excessive formation of advanced glycation end-products (AGEs) presents the most important mechanism of metabolic memory that underlies the pathophysiology of chronic diabetic complications. Independent of the level of hyperglycaemia, AGEs mediate intracellular glycation of the mitochondrial respiratory chain proteins leading to excessive production of reactive oxygen species (ROS) and amplification of their formation. Additionally, AGEs trigger intracellular damage via activation of the receptor for AGEs (RAGE) signalling axis that leads to elevation of cytosolic ROS, nuclear factor kappaB (NF-κB) activation, increased expression of adhesion molecules and cytokines, induction of oxidative and endoplasmic reticulum stress. Recent studies have identified novel microRNAs (miRNAs) involved in the regulation of AGE/RAGE signalling in the context of diabetic micro- and macrovascular complications. The aim of this review is to discuss the emerging role of miRNAs on AGE/RAGE pathway and the potential use of several miRNAs as novel therapeutic targets.

Advanced glycation end products accumulate in the ovarian granulosa-cell layer of women with polycystic ovarian syndrome. Taken that the MAPK/ERK-pathway is a key regulator of oocyte maturation and function, consisting the main pathway used by the gonadotrophic hormones (luteinizing hormone, follicle stimulating hormone) to control ovulation, the present study aims to assess advanced glycation end products' interference into luteinizing hormone-and follicle stimulating hormone-signaling via the MAPK/ERK-pathway in the human granulosa KGN cell line. KGN cells were treated with luteinizing hormone or follicle stimulating hormone in the absence or presence of human glycated albumin. The specific activation of the main components of the MAPK/ERK1/2-pathway (namely c-Raf, MEK and ERK1/2) was assessed. Treatment of KGN cells with an MEK1/2-inhibitor or a blocking anti-RAGE-antibody was also performed to shed further light on the mechanism of the involvement of advanced glycation end products in luteinizing hormone and/or follicle stimulating hormone-related signaling pathways. Luteinizing hormone treatment increased p-ERK1/2 levels in human granulosa cells, while the combined treatment of luteinizing hormone and human glycated albumin provoked a decrease of p-ERK1/2 levels. A similar reducing effect was also observed for the upstream molecule phospho-cRaf upon combined treatment, while treatment with an MEK-inhibitor confirmed that the phenomenon is MAPK/ERK-pathway-dependent. Similarly, follicle stimulating hormone treatment increased p-ERK1/2 and p-MEK1/2 levels, while the combined treatment of follicle stimulating hormone and human glycated albumin downregulated their levels. Advanced glycation end products reduce the luteinizing hormone- and follicle stimulating hormone-induced ERK1/2 activation that is critical for granulosa cell mitogenesis and proliferation. Inappropriate activation of ERK1/2 in granulosa cells may block the granulosa cell differentiation pathway and/or impair follicular responses to hormones, potentially leading to ovulation failure that characterizes polycystic ovarian syndrome.

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT-PCR, semi-quantitative and quantitative real-time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer-cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Student's t-test. HT29 human xenografts were treated with anti-PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non-parametric tests, Kaplan-Meier curves, log-rank test and Cox model. All statistical tests were two-sided. PC1 overexpression promotes epithelial-to-mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence-free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.

Deregulated RTK/RAS/RAF/MEK/ERK signaling (MAPK signaling) drives growth of over 40% of human tumors, most commonly due to activating mutations of pathway components such as in RAS and BRAF genes. Targeting MAPK signaling using RAF and MEK inhibitors elicited remarkable responses and prolonged survival of melanoma patients with BRAF(V600E) mutation, it however showed limited efficacy in colorectal and thyroid BRAF(V600E) cancers. Similarly, MEK inhibitors showed modest effect in other MAPK-dependent tumors, such as RAS-mutant tumors or in Triple-Negative Breast Cancers (TNBC). We and others have shown that relief of negative-feedback upon RAF or MEK inhibitor treatment promotes upregulation of various Receptor Tyrosine Kinases (RTKs), which in turn activate RAS, resulting in rebound of ERK activity in the presence of the inhibitor. Individual targeting of RTKs in combination with RAF or MEK inhibitors is challenging, due to variability in RTK expression in the same tumor as well as across different tumors. SHP2 (PTPN11) is a non-receptor tyrosine phosphatase and scaffold protein that mediates signal transduction downstream of multiple RTKs promoting RAS activation. The recent development of selective small-molecule SHP2 inhibitors provides the opportunity to overcome adaptive resistance by co-targeting MAPK signaling and feedback-induced RTK-mediated RAS activation. By using the allosteric SHP2 inhibitor SHP099, we found that combinatorial targeting of SHP2 and components of MAPK signaling was broadly effective in TNBC models and molecularly defined subsets of RAS and BRAF-mutant tumors. Notably, in each MAPK-driven tumor analyzed, p(Y542)SHP2 induction in response to MAPK signaling inhibition was required for combined treatment sensitivity. In RAS-mutant tumors, sensitivity to combined SHP2 and MEK inhibition was confined to RAS(G12X) tumors, in which mutant RAS depends on RTK-mediated nucleotide exchange for its activity. In tumors expressing RAS(G13D) or RAS(Q61X) the combination was largely ineffective. We further identified BRAF(V600E) colorectal and thyroid tumors in which feedback reactivation of ERK is driven by ERBB or MET receptors and is therefore mediated by SHP2 ("SHP2-positive" tumors). In such tumors, mostly colorectal, the combination of RAF and MEK inhibitors with SHP099 showed dramatic inhibition of MAPK signaling and tumor growth, both ex vivo and in vivo. Finally, we identified a subset of BRAF(V600E) tumors, mostly thyroid, that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Collectively, our findings identify molecular determinants and predictive biomarkers of response to combined SHP2/MAPK signaling inhibition, providing a roadmap for the clinical development of this combinatorial treatment strategy for MAPK-dependent tumors, such as TNBC, and large subsets of RAS-mutant and BRAF-mutant tumors, for which there are no currently available targeted therapeutic options. Citation Format: Christos Adamopoulos, Tamer A. Ahmed, Zoi Karoulia, Xuewei Wu, Ravi Sachidanandam, Stuart A. Aaronson, Poulikos I. Poulikakos. SHP2 drives adaptive resistance to ERK signaling inhibition in molecularly defined subsets of ERK-dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-119.

Atherosclerosis, the principal cause of cardiovascular diseases (CVDs), is a process that involves a complex interplay among different factors and cell types, including cells of the immune system (T cells, B cells, natural killer cells, monocytes/macrophages, dendritic cells) and cells of the vessel wall (endothelial cells [ECs], vascular smooth muscle cells [VSMCs]). The atherogenic process evolves in different stages, starting from inflammatory endothelial activation/dysfunction and resulting in plaque vulnerability and rupture.1 Several cardiovascular risk factors have been recognized. Among them, vitamin D deficiency [25(OH)D