Ribosomal Protein L9 Maintains Stemness of Colorectal Cancer via an ID-1 Dependent Mechanism.

The identification of therapeutic target genes that are functionally involved in stemness is crucial to effectively cure patients with metastatic carcinoma. We have previously reported that inhibition of ribosomal protein L9 (RPL9) expression suppresses the growth of colorectal cancer (CRC) cells by inactivating the inhibitor of DNA-binding 1 (ID-1) signaling axis, which is functionally associated with cancer cell survival. In addition to cell proliferation, ID-1 is also involved in the maintenance of cancer stemness. Thus, we aimed in this study to investigate whether the function of RPL9 could correlate with CRC stem cell-like properties. Here, we demonstrated that siRNA silencing of RPL9 reduced the invasiveness and migrative capabilities of HT29 and HCT116 parental cell populations and the capacity for sphere formation in the HT29 parental cell population. CD133+ cancer stem cells (CSCs) were then separated from CD133- cancer cells of the HT29 parental cell culture and treated with RPL9-specific siRNAs to verify the effects of RPL9 targeting on stemness. As a result, knockdown of RPL9 significantly suppressed the proliferative potential of CD133+ colorectal CSCs, accompanied by a reduction in CD133, ID-1, and p-IκBα levels. In line with these molecular alterations, targeting RPL9 inhibited the invasion, migration, and sphere-forming capacity of CD133+ HT29 CSCs. Taken together, these findings suggest that RPL9 promotes CRC stemness via ID-1 and that RPL9 could be a potential therapeutic target for both primary CRC treatment and the prevention of metastasis and/or recurrence.

Site-specific mutagenesis screening in KRASG12D mutant library to uncover resistance mechanisms to KRASG12D inhibitors.

KRAS plays a crucial role in regulating cell survival and proliferation and is one of the most commonly mutated oncogenes in human cancers. The novel KRASG12D inhibitor, MRTX1133, demonstrates promising antitumor efficacy in vitro and in vivo. However, the development of acquired resistance in treated patients presents a considerable challenge to sustained therapeutic effectiveness. In response to this challenge, we conducted site-specific mutagenesis screening to identify potential secondary mutations that could induce resistance to MRTX1133. We screened a range of KRASG12D variants harboring potential secondary mutations, and 44 representative variants were selected for in-depth validation of the pooled screening outcomes. We identified eight variants (G12D with V9E, V9W, V9Q, G13P, T58Y, R68G, Y96W, and Q99L) that exhibited substantial resistance, with V9W showing notable resistance, and downstream signaling analyses and structural modeling were conducted. We observed that secondary mutations in KRASG12D can lead to acquired resistance to MRTX1133 and BI-2865, a novel pan-KRAS inhibitor, in human cancer cell lines. This evidence is critical for devising new strategies to counteract resistance mechanisms and, ultimately, enhance treatment outcomes in patients with KRASG12D-mutant cancers.

Targeted kinase degradation via the KLHDC2 ubiquitin E3 ligase.

Chemically induced protein degradation is a powerful strategy for perturbing cellular biochemistry. The predominant mechanism of action for protein degrader drugs involves an induced proximity between the cellular ubiquitin-conjugation machinery and a target. Unlike traditional small molecule enzyme inhibition, targeted protein degradation can clear an undesired protein from cells. We demonstrate here the use of peptide ligands for Kelch-like homology domain-containing protein 2 (KLHDC2), a substrate adapter protein and member of the cullin-2 (CUL2) ubiquitin ligase complex, for targeted protein degradation. Peptide-based bivalent compounds that can induce proximity between KLHDC2 and target proteins cause degradation of the targeted factors. The cellular activity of these compounds depends on KLHDC2 binding. This work demonstrates the utility of KLHDC2 for targeted protein degradation and exemplifies a strategy for the rational design of peptide-based ligands useful for this purpose.

RPL27 contributes to colorectal cancer proliferation and stemness via PLK1 signaling.

Although expression of ribosomal protein L27 (RPL27) is upregulated in clinical colorectal cancer (CRC) tissue, to the best of our knowledge, the oncogenic role of RPL27 has not yet been defined. The present study aimed to investigate whether targeting RPL27 could alter CRC progression and determine whether RPL27 gains an extra­ribosomal function during CRC development. Human CRC cell lines HCT116 and HT29 were transfected with RPL27­specific small interfering RNA and proliferation was assessed in vitro and in vivo using proliferation assays, fluorescence­activated cell sorting (FACS) and a xenograft mouse model. Furthermore, RNA sequencing, bioinformatic analysis and western blotting were conducted to explore the underlying mechanisms responsible for RPL27 silencing­induced CRC phenotypical changes. Inhibiting RPL27 expression suppressed CRC cell proliferation and cell cycle progression and induced apoptotic cell death. Targeting RPL27 significantly inhibited growth of human CRC xenografts in nude mice. Notably, polo­like kinase 1 (PLK1), which serves an important role in mitotic cell cycle progression and stemness, was downregulated in both HCT116 and HT29 cells following RPL27 silencing. RPL27 silencing reduced the levels of PLK1 protein and G2/M­associated regulators such as phosphorylated cell division cycle 25C, CDK1 and cyclin B1. Silencing of RPL27 reduced the migration and invasion abilities and sphere­forming capacity of the parental CRC cell population. In terms of phenotypical changes in cancer stem cells (CSCs), RPL27 silencing suppressed the sphere­forming capacity of the isolated CD133+ CSC population, which was accompanied by decreased CD133 and PLK1 levels. Taken together, these findings indicated that RPL27 contributed to the promotion of CRC proliferation and stemness via PLK1 signaling and RPL27 may be a useful target in a next­generation therapeutic strategy for both primary CRC treatment and metastasis prevention.

RPL17 Promotes Colorectal Cancer Proliferation and Stemness through ERK and NEK2/ß-catenin Signaling Pathways.

Aims: Ribosomal protein L17 (RPL17), a 60S subunit component, is up-regulated in colorectal cancer (CRC). However, its oncogenic role in CRC progression remains unexplored. Thus, we aimed to investigate the effect of RPL17 targeting on CRC in vitro and in vivo and whether RPL17 gained an extra-ribosomal function during CRC development. Methods: RPL17-specific siRNAs complexed with cationic lipids were transfected to CRC cells to silence target gene expression and then real-time RT-PCR and western blotting were applied to observe the change of expression or activity of genes or proteins of interest. Cell proliferation assay, clonogenic assay and cell cycle analysis were used to determine the in vitro effects of RPL17siRNAs on CRC cell growth, and a subcutaneous xenograft assay was applied to test the effect of RPL17siRNAs on in vivo tumor growth. RNA sequencing and western blotting were used to investigate the underlying mechanisms. Sphere-forming assay, invasion assay and migration assay were used to evaluate the effects of RPL17siRNAs on CRC stemness. Results: siRNA-mediated inhibition of RPL17 expression suppressed CRC cell growth and long-term colony formation by inducing apoptotic cell death. Similarly, targeting RPL17 effectively suppressed tumor formation in a mouse xenograft model. RNA sequencing of RPL17-silenced CRC cells revealed the same directional regulation of 159 (93 down- and 66 up-regulated) genes. Notably, NIMA-related kinase 2 (NEK2), which functionally cooperates with extracellular-regulated protein kinase (ERK) and plays a pivotal role in mitotic progression and stemness maintenance, was down-regulated. RPL17 silencing reduced NEK2, ß-catenin, and p-ERK protein levels. These molecular alterations reflected the reduction in sphere-forming capacity, expression of stem cell marker genes, migration, and invasion. Reversely, RPL17 overexpression increased the ability of long-term colony formation, migration, and invasion. Conclusion: Our findings indicate that RPL17 promotes CRC proliferation and stemness via the ERK and NEK2/ß-catenin signaling axis, and targeting RPL17 could be the next molecular strategy for both primary CRC treatment and prevention of secondary tumor formation.

Identification of Pyridinyltriazine Derivatives as Potent panFGFR Inhibitors against Gatekeeper Mutants for Overcoming Drug Resistance.

Although FGFR inhibitors hold promise in treating various cancers, resistance to the FGFR inhibitors caused by acquired secondary mutations has emerged. To discover novel FGFR inhibitors capable of inhibiting FGFR mutations, including gatekeeper mutations, we designed and synthesized several new pyridinyltriazine derivatives. A structure-activity relationship (SAR) study led to the identification of 17a as a highly potent panFGFR inhibitor against wild-type and mutant FGFRs. Notably, 17a is superior to infigratinib in terms of kinase-inhibitory and cellular activities, especially against V555M-FGFR3. Molecular dynamics simulations provide a clear understanding of why pyridinyltraizine derivative 17a possesses activity against V555M-FGFR3. Moreover, 17a significantly suppresses proliferation of cancer cells harboring FGFR mutations via FGFR signaling blockade, cell cycle arrest, and apoptosis. Furthermore, 17a and 17b exhibited remarkable efficacies in TEL-V555M-FGFR3 Ba/F3 xenograft mouse model and 17a is more efficacious than infigratinib. This study provides new insight into the design of novel FGFR inhibitors that are active against FGFR mutants.

Complete Genome Sequence of Stenotrophomonas maltophilia Podophage Paxi.

Stenotrophomonas maltophilia is a multidrug-resistant nosocomial pathogen that can cause life-threatening infections among immunocompromised populations. This report presents the complete 74,962-bp genome of S. maltophilia podophage Paxi, an N4-like phage sharing 85.3% nucleotide similarity to S. maltophilia podophage Pokken.

Novel Small Molecules Capable of Blocking mtRAS-Signaling Pathway.

RAS mutants are involved in approximately 30% of all human cancers and have been regarded as undruggable targets owing to relatively smooth protein surface and obscure binding pockets. In our previous study, we have demonstrated that GNF-7, a multi-targeted kinase inhibitor, possesses potent anti-proliferative activity against Ba/F3 cells transformed with NRAS-G12D. Based on our further analysis using Ba/F3 cells transformed with mtRAS, we discovered a series of pyrimido[4,5-d]pyrimidin-2-one analogues as mtRAS-signaling pathway blockers. In addition, our efforts expanded the assessment to cancer cells with mtRAS, which revealed that these substances are also capable of strongly suppressing the proliferation of various cancer cells harboring KRAS-G12D (AsPC-1), KRAS-G12V (SW480, DU-145), KRAS-G12C (H358), KRAS-G13D (MDA-MB-231), KRAS-Q61L (HT-29), and NRAS-Q61L (OCI-AML3). We herein report novel and potent mtRAS-signaling pathway blockers, SIJ1795 and SIJ1772, possessing 2 to 10-fold increased anti-proliferative activities compared to those of GNF-7 on cancer cells harboring mtRAS as well as on Ba/F3 cells transformed with mtRAS. Both SIJ1795 and SIJ1772 attenuate phosphorylation of RAS downstream molecules (AKT and MEK) and induce apoptosis and G0/G1 cell cycle arrest on cancer cells with mtRAS. Moreover, both substances substantially suppress the migration, invasion, and colony formation of cancer cells harboring mtRAS. Taken together, this study led us to identification of SIJ1795 and SIJ1772 capable of strongly inhibiting mtRAS-signaling pathway on cancer cells harboring mtRAS.

Identification of Thieno[3,2-d]pyrimidine Derivatives as Dual Inhibitors of Focal Adhesion Kinase and FMS-like Tyrosine Kinase 3.

Focal adhesion kinase (FAK) is overexpressed in highly invasive and metastatic cancers. To identify novel FAK inhibitors, we designed and synthesized various thieno[3,2-d]pyrimidine derivatives. An intensive structure-activity relationship (SAR) study led to the identification of 26 as a lead. Moreover, 26, a multitargeted kinase inhibitor, possesses excellent potencies against FLT3 mutants as well as FAK. Gratifyingly, 26 remarkably inhibits recalcitrant FLT3 mutants, including F691L, that cause drug resistance. Importantly, 26 is superior to PF-562271 in terms of apoptosis induction, anchorage-independent growth inhibition, and tumor burden reduction in the MDA-MB-231 xenograft mouse model. Also, 26 causes regression of tumor growth in the MV4-11 xenograft mouse model, indicating that it could be effective against acute myeloid leukemia (AML). Finally, in an orthotopic mouse model using MDA-MB-231, 26 remarkably prevents metastasis of orthotopic tumors to lymph nodes. Taken together, the results indicate that 26 possesses potential therapeutic value against highly invasive cancers and relapsed AML.

Novel and Potent Small Molecules against Melanoma Harboring BRAF Class I/II/III Mutants for Overcoming Drug Resistance.

Melanoma accounts for the majority of skin cancer deaths. About 50% of all melanomas are associated with BRAF mutations. BRAF mutations are classified into three classes with regard to dependency on RAF dimerization and RAS signaling. The most frequently occurring class I BRAF V600 mutations are sensitive to vemurafenib whereas class II and class III mutants, non-V600 BRAF mutants are resistant to vemurafenib. Herein we report six pyrimido[4,5-d]pyrimidin-2-one derivatives possessing highly potent anti-proliferative activities on melanoma cells harboring BRAF class I/II/III mutants. Novel and most potent derivative, SIJ1777, possesses not only two-digit nanomolar potency but also 2 to 14-fold enhanced anti-proliferative activities compared with reference compound, GNF-7 against melanoma cells (SK-MEL-2, SK-MEL-28, A375, WM3670, WM3629). Moreover, SIJ1777 substantially inhibits the activation of MEK, ERK, and AKT and remarkably induces apoptosis and significantly blocks migration, invasion, and anchorage-independent growth of melanoma cells harboring BRAF class I/II/II mutations while both vemurafenib and PLX8394 have little to no effects on melanoma cells expressing BRAF class II/III mutations. Taken together, our six GNF-7 derivatives exhibit highly potent activities against melanoma cells harboring class I/II/III BRAF mutations compared with vemurafenib as well as PLX8394.