Artigos de revistas sobre o tema "Kd 3310"

The chemical composition of shaddock mainly includes polyphenols, proteins and polysaccharides. However, polysaccharides from shaddock materials have received much less consideration than polyphenols (Fellers et al., 1990). Herein the chemical compositions, ?-glucosidase and ?-amylase inhibitory activities of crude polysaccharides from the endodermis of shaddock were investigated. The exopolysaccharides (EPS) exhibited a broad and intense peak at 3300-3400 cm-1 that characterized the absorption of the hydroxyl group, and one weak C-H band at around 2941.3 cm-1 in the IR spectrum. The content of neutral sugars in EPS was determined as 37.16%. The content of acidic sugar in EPS was determined as 33.71%. EPS exhibited the highest content of neutral sugar. The content of proteins in EPS was 5.75%. The content of polyphenols was 6.52%. The EPS mainly consisted of four types of polysaccharides with molecular weights of 110 kD, 68 kD, 31 kD and 12 kD. The crude EPS showed significantly higher inhibitory effects on ?-glucosidase and ?-amylase (inhibition to 74.12% and 86.59%, respectively).

Rhyolites and basite rocks are present in the Archaean greenstone belts of the Kursk Domain (KD) of the East Sarmatia. The rhyolite age is 3122 ± 9 Ma (zircons, SIMS). A positive εNd (3122) = + 0.9 for rhyolites and their Sm-Nd model age ТNd (DM) = 3300 Ma as well as the age of the inherited zircon (3250 Ma) testifies to the participation of the more ancient crust component in the formation of rhyolite magmas. In geochemistry, rhyolites are very close to the TTG of the KD with an age 2.96-3.03 Ga. In the Middle Dnieper granite - greenstone area there are rhyolites and dacites with an age of 3.12 Ga with εNd (T) = + 0.6 - (+1.2) and very close geochemical characteristics. Thus, the hypothesis of a common geological history of the eastern part of Ukrainian Shield and KD in Mesoarchean is confirmed.

In order to understand the sorption behavior of 137Cs and 90Sr into soil sample from Rembang and Subang, it is important to estimate the effect of contact time, ionic strength and concentration of metal ion in the solution. For this reason, the interaction of 137Cs and 90Sr with soil sample has been examined. The study performed at trace concentration (~10-8 M) of CsCl and SrCl2, and batch method was used. NaCl has been selected as a representative of the ionic strength with 0.1; 0.5 and 1.0 M concentrations. Concentration of 10-8~10-4 M CsCl and SrCl2 were used for study the effect of Cs and Sr concentrations in solution. Apparent distribution coefficient was used to predict the sorption behavior. The sorption equilibrium of 137Cs and 90Sr into soil was attained after 5 days contacted with Kd value around 3300-4200 mL/g, where Kd was defined as the ratio of number of radionuclide activity absorbed in solid phase per-unit mass to the number of radionuclide activity remains is solution per-unit volume. Presence of NaCl as background salt in the solution affected Kd values due to competition among metal ions into soil samples. Increase of Cs or Sr concentration in solution made Kd value decreased drastically. This information is expected could provide an important input for the planning and design of radioactive waste disposal system in Java Island in the future.

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively, and less commonly in other cancers. We previously demonstrated that LY3962673 is a highly potent inhibitor of KRAS G12D and is selective against wild-type (WT) KRAS in mutant -cell lines and -in vivo models. Here, we describe the mechanism by which LY3962673 inhibits KRAS G12D and report a more comprehensive evaluation of LY3962673 activity across a panel of genetically and histologically diverse cancer cell lines, as well as in multiple patient-derived xenograft (PDX) models. LY3962673 is a non-covalent KRAS G12D inhibitor with high affinity binding to KRAS G12D-GDP (Kd 0.071 nM) compared to KRAS G12D-GTPγS (Kd 26.7 nM). In a panel of cancer cell lines with KRAS G12D mutations, non-G12D mutations or KRAS WT, LY3962673 selectively suppressed MAPK signaling and inhibited the growth of KRAS G12D mutant cancer cells while sparing KRAS WT and non-G12D mutant cells. Sensitivity to LY3962673 varied among the KRAS G12D-mutant cells tested, suggesting that not all cell lines share the same dependence on KRAS G12D for their growth and survival. Furthermore, in multiple KRAS G12D-mutant PDX models representing diverse tumor types, LY3962673 demonstrated anti-tumor activities, ranging from tumor growth inhibition to robust tumor regression. LY3962673 also showed enhanced efficacy when combined with other anti-cancer agents. Taken together, the findings underscore the potential of LY3962673 as a monotherapy or in combination with other anti-cancer agents, as a promising oral therapeutic option for a range of cancer types with KRAS G12D mutations. Citation Format: Xueqian Gong, Hong Gao, Mark H. Bender, Wenyu Ming, Youyan Zhang, Trent R. Stewart, Chun Ping Yu, Wei Guo Xu, Aurthur Xintian You, Wen Ting Bian, Binghui Li, Tao Wang, Huimin Bian, Manuj Tandon, Andrew Capen, Rachel N. Cavitt, Bryan D. Anderson, Wayne Bocchinfuso, Anke Klippel, Chandrasekar Iyer. LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3316.

Introduction: Achievement of minimal residual disease negative (MRD-) status in multiple myeloma (MM) is associated with improved progression-free survival (PFS) and overall survival (OS). Isatuximab (Isa) is an approved anti-CD38 IgG kappa monoclonal antibody. We analyzed the depth of response including MRD-, long-term outcomes, and kinetics of tumor response in the IKEMA study. Measurement by mass spectrometry of serum M-protein was also performed to overcome the interference with Isa in standard immunofixation assay. Methods: IKEMA was a randomized, open-label, multicenter Phase 3 study that investigated Isa plus carfilzomib and dexamethasone (Isa-Kd) vs Kd in patients (pts) with relapsed MM who received 1-3 lines of therapy (NCT03275285). The primary endpoint of PFS and secondary endpoints of overall response rate (ORR), very good partial response or better (≥VGPR) and complete response (CR) rate were determined by an Independent Response Committee based on central data for M-protein, central imaging review and local bone marrow for plasma cell infiltration according to IMWG criteria.MRD was assessed in bone marrow aspirates from pts who achieved ≥VGPR by next generation sequencing at 10-5 sensitivity level. Mass spectrometry analysis was performed to measure serum M-protein without Isa interference. Hazard ratios and corresponding confidences interval were estimated using Cox proportional hazards model. Secondary endpoints were compared between treatment arms using Cochran Mantel Haenszel test. Per ITT, all randomized pts not reaching MRD- or without MRD assessment were analyzed as MRD+. Results: 302 pts (179 Isa-Kd, 123 Kd) were randomized. At a median follow-up of 20.7 months deeper responses were observed in Isa-Kd vs Kd with ≥VGPR 72.6% vs 56.1% (nominal p=0.011) and ≥CR 39.7% vs 27.6%, respectively. MRD- occurred in 53/179 (30%) pts in the Isa-Kd arm vs 16/123 (13%) in the Kd arm (nominal p=0.0004) with 20.1% (36/179 pts Isa-Kd) vs 10.6% (13/123 pts Kd) reaching CR and MRD-. PFS by MRD status is shown in the Figure, HR favors Isa-Kd vs Kd in both MRD- pts (HR 0.578, 95%CI: 0.052-6.405) and MRD+ pts (HR 0.670, 95% CI: 0.452-0.993). MRD- pts had a longer PFS than MRD+ pts. Within Isa-Kd, MRD-negative status could be obtained in pts with renal impairment (26.5% MRD- vs 25.9% MRD+ with eGFR <60mL/min/1.73 m2); with ISS stage III at diagnosis (32.1% MRD- vs 27.8% MRD+); with t(4;14) [13.2% MRD- vs 11.9% MRD+], with gain(1q21) [45.3% MRD- vs 40.5% MRD+]; in heavily pretreated ≥3 prior lines (22.6% MRD- vs 19.0% MRD+) or refractory to lenalidomide in last regimen (18.9% MRD- vs 20.6% MRD+). Within Isa-Kd, MRD-negative status was reached less frequently in pts refractory to a proteasome inhibitor (PI) [18.9% MRD-vs 36.5% MRD+) or with del(17p), [3.8% MRD- vs 12.7% MRD+]. Interference of Isa with M protein was explored: samples from 27 pts with near-CR (only serum immunofixation (IF) positive IgG kappa) or potential CR (serum remaining M-protein ≤ 0.5 g/dL with IF positive IgG kappa) in the Isa-Kd arm were tested by mass spectrometry. Among them, 11 near CR or potential CR pts had documented <5% plasma cells in bone marrow and were mass spectrometry negative (residual myeloma M-protein level below LOQ of central lab immunofixation). In addition, 7/11 were also MRD-. These results support that both current CR rate and MRD- CR rate are underestimated (potential adjusted CR rate of 45.8%; potential adjusted MRD- CR rate 24%). Responses occurred quickly in both arms. The median time (Isa-Kd vs Kd) in responders to: first response was 32.0 (28-259) days vs 33.0 (27-251) days; best response 120.0 (29-568) days vs 104.5 (29-507) days; first CR 184.0 (30-568) days vs 229.5 (58-507) days; first ≥VGPR 88.0 (28-432) vs 90.0 (29-491) days, respectively. In addition to increased depth of response, quality of life as measured by QLQ-C30 Global Health Status scores was maintained with Isa-Kd per descriptive analyses. Conclusions: There was a clinically meaningful improvement in depth of response in Isa-Kd vs Kd. CR rate in Isa-Kd of 39.7% was underestimated due to interference. Mass spectrometry results suggest that the potential adjusted CR rate could be reached for 45.8% of pts with 1 to 3 prior lines treated in Isa-Kd. More pts in Isa-Kd vs Kd reached MRD negativity (30% vs 13%) and at least twice as many reached CR MRD- (20.1% vs 10.6%; adjusted 24% vs 10.6%). Reaching MRD negativity was associated with longer PFS in both arms. Disclosures Martin: Legend Biotech: Consultancy; Sanofi, Amgen, Seattle Genetics, JNJ - Janssen: Research Funding. Mikhael:Amgen, Celgene, GSK, Janssen, Karyopharm, Sanofi, Takeda: Honoraria. Hajek:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Kim:Amgen, BMS, Janssen, Sanofi, Takeda: Consultancy, Honoraria, Research Funding. Suzuki:Takeda, Celgene, ONO, Amgen, Novartis, Sanofi, Bristol-Myers Squibb, AbbVie and Janssen: Honoraria; Bristol-Myers Squibb, Celgene and Amgen: Research Funding; Takeda, Amgen, Janssen and Celgene: Consultancy. Hulin:Celgene/Bristol-Myers Squibb, Janssen, GlaxoSmithKline, and Takeda: Honoraria. Garg:Janssen, Takeda, Celgene, Novartis, Sanofi: Honoraria. Quach:GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Consultancy; GlaxoSmithKline, Karyopharm, Amgen, Celgene, Janssen Cilag: Honoraria; Amgen, Celgene, karyopharm, GSK, Janssen Cilag, Sanofi.: Membership on an entity's Board of Directors or advisory committees; Amgen, sanofi, celgene, Karyopharm, GSK: Research Funding. Risse:Sanofi: Current Employment. Asset:Sanofi: Current Employment. Macé:Sanofi: Current Employment. van de Velde:Sanofi: Current Employment, Current equity holder in publicly-traded company. Moreau:Janssen: Consultancy, Honoraria; Novartis: Honoraria; Takeda: Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. OffLabel Disclosure: Isatuximab, a monoclonal CD38 antibody, is approved in combination with pomalidomide and dexamethasone in the United States, the European Union, Canada, Australia, Switzerland, and Japan for the treatment of adult patients with relapsed/refractory multiple myeloma who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor.

Introduction: Isatuximab (Isa) is an immunoglobulin (Ig)G1 monoclonal antibody targeting a CD38 transmembrane glycoprotein in multiple myeloma (MM). Isa is approved for use in multiple countries to treat adults with relapsed/refractory MM (RRMM) when given in combination with either pomalidomide-dexamethasone (Pd) or carfilzomib-dexamethasone (Kd). Hypogammaglobulinemia (HGG; polyclonal IgG <4 g/L) and the associated infection risk is a potential complication for patients (pts) with MM treated with immunotherapies. High infection and HGG rates have been reported during treatment (tx) with antimyeloma therapies, and in particular, recently, with anti-B cell maturation antigen-directed therapies. 1,2 Here, we explore the impact of Isa monotherapy on Ig levels, and of approved Isa combinations (Isa-Pd and Isa-Kd) on Ig levels and the incidence, severity, and clinical significance of HGG, including the incidence of infections depending on concomitance of HGG or not, in pts with RRMM. Methods: Pts with RRMM with central lab Ig data from 3 monotherapy studies were pooled (across dose levels, most >10 mg/kg) to retrospectively explore Ig levels throughout tx with Isa monotherapy until disease progression, intolerable toxicity, or withdrawal.We performed a side-by-side analysis of pts from the Phase 3 ICARIA-MM (Isa-Pd vs Pd) and IKEMA (Isa-Kd vs Kd) studies with/without HGG at baseline and concomitance of HGG with infections. Pts were analyzed overall and according to the heavy chain of the monoclonal protein at study entry, where IgG consisted of monoclonal IgG (M-spike in g/dL *1000) subtracted from total quantitative IgG (mg/dL) to obtain the true polyclonal IgG level, and non-IgG consisted of other Ig or light chain subtypes. Recovery was defined as >6 g/L (complete) or ≥4 g/L (partial) after having <4 g/L at any time. Results: Isa monotherapy analyses revealed a fast decrease in non-involved IgA levels after 1 month, followed by stabilization, which has also been reported with daratumumab monotherapy. Isa monotherapy did not induce decreased IgG levels. In ICARIA-MM, more pts with non-IgG vs IgG MM at study entry had HGG during tx (54.0% vs 14.4%, Isa-Pd; 39.6% vs 12.7%, Pd), whereas incidences were similar in IKEMA (11.7% vs 8.9%, Isa-Kd; 15.5% vs 11.4%, Kd). A smaller proportion of pts with HGG vs without HGG at any time during the study had definitive tx discontinuation in both ICARIA-MM and IKEMA; discontinuations related to adverse events (AEs) were less frequent in pts with HGG vs without HGG (Table). Of these AEs, infections led to discontinuation in 33.3% to 46.2% of patients with HGG at any time, regardless of tx (Table). In ICARIA-MM, 70.9% (Isa-Pd) and 55.5% (Pd) of pts without HGG at baseline developed HGG post-baseline; in IKEMA, 90.6% (Isa-Kd) and 82.0% (Kd) developed HGG post-baseline. Among pts who received Isa-Pd, those who had IgG MM at study entry experienced a larger proportion of recovery (9.1%, complete; 26.0%, partial) than those with non-IgG MM (6.8%, complete; 15.9%, partial); similar results were seen with Pd in pts with (21.9%, complete; 40.6%, partial) and without IgG MM (4.8%, complete; 19.0%, partial). Pts who had IgG MM at study entry and received Isa-Kd experienced a larger proportion of recovery (17.0%, complete; 33.0%, partial) than those with non-IgG MM (3.8%, complete; 7.5%, partial); similar results were seen with Kd in pts with (14.7%, complete; 25.3%, partial) and without IgG MM (3.1%, complete; 3.1%, partial). Infections of any grade concomitant with HGG were reported in 28.2% of pts in ICARIA-MM and 59.2% in IKEMA, with a larger proportion of pts receiving Isa combinations reporting infections of any grade and Grade ≥3 (Table). Respiratory infections of any grade concomitant with HGG were more frequent in pts receiving Isa-Pd vs Pd and Isa-Kd vs Kd (Table). There were low proportions of pts with Grade ≥3 respiratory infections concomitant with HGG regardless of tx arm (Table). Conclusions: Monotherapy with Isa had little impact on Ig levels. Among pts treated with Isa monotherapy, there is an early drop in non-involved IgA levels, followed by stabilization. In ICARIA-MM and IKEMA, HGG did not contribute to increased Grade ≥3 infections. Isa added to backbone tx is safe with a low rate of Grade ≥3 infections despite the decrease in Ig levels. Funding: Sanofi. References: 1. Lancman G, et al. Blood. 2022;140:10073-4. 2. Rodriguez-Otero P, et al. J Clin Oncol. 2023;41:8020.

Chemical modification experiments with tetranitromethane (TNM) have been used to investigate the role of tyrosine residues in the formation of the complex between PpL (the single Ig-binding domain of protein L, isolated from P. magnus strain 3316) and the kappa light chain (κ-chain). Reaction of PpL with TNM causes the modification of 1.9 equiv. of tyrosine (Tyr51 and Tyr53) and results in an approx. 140-fold decrease in affinity for human IgG. Similar experiments with mutated PpL proteins suggest that nitration predominantly inactivates the protein by modification of Tyr53. Reduction of the nitrotyrosine groups to aminotyrosine by incubation with sodium hydrosulphite does not restore high affinity for IgG. Modification of κ-chain by TNM resulted in the nitration of 3.1±0.09 tyrosine residues. When the PpLŐκ-chain complex was incubated with TNM, 4.1±0.04 tyrosine residues were nitrated, indicating that one tyrosine residue previously modified by the reagent was protected from TNM when the proteins are in complex with each other. The Kd for the equilibrium between PpL, human IgG and their complex has been shown by ELISA to be 112±20nM. A similar value (153±33nM) was obtained for the complex formed between IgG and the Tyr64 → Trp mutant (Y64W). However, the Kd values for the equilibria involving the PpL mutants Y53F and Y53F,Y64W were found to be 3.2±0.2 and 4.6±1µM respectively. These suggest that the phenol group of Tyr53 in PpL is important to the stability of the PpLŐκ-chain complex.

Abstract Background: KRAS mutations are the most frequently encountered driver oncogene, involved in ~25% of all human cancers [1,2]. KRASG12D is the predominant KRAS mutation isoform, detected in approximately 35% of pancreatic cancer, 13% of colorectal cancer, and 5% of NSCLC [3]. Compared to KRASG12C, targeting KRASG12D has proven to be more challenging since the target protein lacks a reactive amino acid residue for irreversible inhibitory modification by a ligand. Herein, we disclose TSN1611, a potent and selective KRASG12D inhibitor, which possesses favorable oral PK profiles and demonstrates significant in vitro and in vivo anti-tumor activity in various KRASG12D-mutant models. Method: Biochemical HTRF assay was used to measure the inhibition of TSN1611 to both GDP-bound and GTP-bound state of KRASG12D. Biophysical SPR method was used to directly measure the binding of TSN1611 to GDP-bound KRASG12D and KRASWT. Cell based activities were evaluated in a series of in vitro cell proliferation assay utilizing Ba/F3 cells engineered with KRASG12D or non-KRASG12D mutations and tumor cell lines harboring KRASG12D mutation. Human cancer cell-derived xenograft models of HPAC (pancreatic) and GP2D (colorectal) were used to evaluate its in vivo antitumor effect. in vitro and in vivo PK studies were performed in mouse, rat, and dog. Systematic nonclinical safety evaluations, including safety panel screen testing, safety pharmacology studies, and repeat-dose toxicity studies were carried out to assess its preliminary toxicity profile. Results: TSN1611 inhibited both active (GTP-bound) and inactive (GDP-bound) forms of KRASG12D protein at IC50 1.23 and 1.49 nM, respectively; the KD value of its direct binding to KRASG12D protein is 1.93 pM in SPR assay. TSN1611 demonstrated potent anti-proliferation activity against several tumor cell lines harboring KRASG12D mutation, and excellent selectivity over cells of NRAS, HRAS, and other KRAS isoforms. It also showed dose-dependent anti-tumor efficacy in GP2D and HPAC models. Mechanism of action studies concluded that the antitumor effect of TSN1611 is resulted from its effective inhibition of KRAS signaling pathway. Oral bioavailability and safety profile across multiple species supported its further development. Conclusion: TSN1611 is a selective KRASG12D inhibitor. It exhibited excellent selectivity and activity both in vitro and in vivo; it demonstrated favorable physicochemical properties, oral PK profiles, and brain penetration potential; it also showed acceptable margins of safety. The preclinical data supports further development. Pending regulatory submission and review, the phase I/II study is planned to start in H1 of 2024. References: [1] Cox, A.D. et al. Nat. Rev. Drug. Discov. 2014, 13, 828. [2] Indini, A. et al. Pharmaceutics 2021, 13, 653. [3] Moore, A. R. et al. Nat. Rev. Drug. Discov. 2020, 19, 533. Citation Format: Erchang Shang, Boyu Zhong, Tony Zhang, Chunlan Dong, Shengtang Ma, Anjiang Yang, Ziyang Jia, Renjuan Zheng, Jing Li, Han Fu, Liangbao Lai. Preclinical studies of TSN1611, a potent, selective, and orally bioavailable KRASG12D inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3315.

Lipoprotein(a) [Lp(a)] is a risk factor for coronary artery disease. It is composed of lipids and apolipoprotein(a) [apo(a)] linked to apolipoprotein B (apoB) by a disulphide bond between Cys-4057 of apo(a)'s kringle 36 and possibly Cys-3734 of apoB. We call this the covalent apo(a): apoB-Lp interaction, to distinguish it from the non-covalent apo(a)/Lp(a): apoB-Lp interaction, which is probably mediated by apo(a)'s kringle 33 and residues 3304-3317 of apoB. The non-covalent interaction could be the initial interaction which brings apo(a) and apoB together prior to covalent linkage and Lp(a) formation. The non-covalent apo(a)/Lp(a)-binding site on apoB is evolutionarily more ancient than the covalent apo(a)-binding site on apoB. Both human and non-human low-density lipoproteins (LDLs) bind non-covalently to human apo(a)/Lp(a); however, only rabbit and human LDLs bind covalently to human apo(a). The non-covalent interaction between mouse LDL and human apo(a)/Lp(a) has a Kd of (1.7 +/- 1.33) x 10(-7) M (n = 3). This explains the co-localization of human apo(a) and mouse apoB in the atherosclerotic lesions of human apo(a) transgenic mice and supports our hypothesis that the non-covalent interaction is a contributing factor to apo(a) atherogenicity.

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between ∼3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type–dependent size differences of ≤70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

Abstract Hematopoietic stem and progenitor cell (HSPC) migration, marrow homing and engraftment are key physiological processes regulating hematopoiesis post transplantation. These processes are the result of the orchestrated actions of multiple extracellular stimuli, which regulate actin remodeling, cell polarity, chemotaxis and cell-cell interactions. In HSPC, the Rho GTPases Rac and CDC42 act as molecular switches that integrate extracellular stimuli in a spatially regulated manner to control cell migration and mediate homing to marrow and mobilization as well as cell survival/ proliferation pathways to mediate engraftment(Gu et al., Science 2003; Cancelas et al., Nature Medicine 2005; Wang et al., Blood 2006). Using an inhibitory peptide against Group A p21 activated kinases (Pak1-3), key effectors of Rac/ CDC42 and individual Pak1 & 2 genetic knock-out mice, we recently demonstrated that Pak kinases, specifically Pak2, are important for HSPC homing and engraftment (Dorrance et al., Blood 2013). Pak2 is a multi-domain protein that contains a C-terminal kinase domain and multiple N-terminal protein-interaction domains. Among these is a non-classical SH3-binding site for the guanine-nucleotide-exchange factor β-PIX, which was shown to be critical for both activation of Rac1 and its localization to and induction of membrane ruffles (Klooster et al., Journal of Cell Biology 2006). In this study we further explored the role of these domains of Pak2 in key HSPC functions, including homing to bone marrow in vivo. We employed a multi-cistronic retrovirus vector that simultaneously deleted floxed endogenous Pak2 gene sequences and rescued with either wild type (WT), a kinase dead (KD) mutant (K278A, defective in auto/ trans phosphorylation) or a Δβ-PIX mutant, (P185/R186A, that cannot bind to β-PIX). As previously demonstrated deletion of Pak2 (Pak2 Δ/Δ) was associated with abnormal SDF-1 stimulated cell protrusions containing F-actin (as demonstrated by confocal and electron microscopy) and these HSPC displayed decreased directional migration (Euclidean distance in Pak2Δ/Δ vs. Pak2WT/WT: 39.6µm ±9.6 vs. 96.6µm ±21.6; P<0.05). This phenotype of abnormal cell protrusions and decreased directional migration was rescued by expressing Pak2-WT (Pak2WT/WT vs. Pak2-WT: 96.6µm ±21.6 vs. 74.0µm ±18.7; P: not significant) but not by expressing Pak2-KD (Pak2WT/WT vs. Pak2-KD: 96.6µm ±21.6 vs. 33.6µm ±6.3; P<0.05) demonstrating the requirement of Pak2 kinase activity in SDF1-induced cell polarization and directed cell migration. Interestingly, we found abnormal F-actin clustering associated with defective polarization (by confocal microscopy) and decreased velocity of cell migration in time-lapsed video microscopy when Pak2-deletion was rescued with Pak2-Δβ-PIX (velocity of migration Pak2WT/WT vs. Pak2-Δβ-PIX, 0.32µm/minute ±0.02 vs. 0.13µm/minute ±0.02; P<0.001), indicating the requirement of β-PIX exchange factor interaction with Pak2 in directed migration. To test whether these in vitro phenotypes were associated with changes in homing efficiency to bone marrow, we performed in vivo homing assays of rescued HSPC. Transduced, GFP-sorted Lin-Sca1+Kit+ cells of each genotype were injected into lethally-irradiated C57BL/6 recipient mice (N= 12-29 /genotype). Twelve hours post-transplantation the number of EGFP+ cells in the bone marrow was determined and percent homing is calculated. Compared to Pak2 WT/WT, Pak2Δ/Δ HSPC displayed reduced homing (99.26%± 4.9 vs. 53.4% ± 4.2; P< 0.0001). The homing defect was rescued by Pak2-WT (Pak2WT/WT vs. Pak2-WT rescue: 99.26%± 4.9 vs. 86% ± 8.5; P: not significant). However neither Pak2-KD nor Pak2-Δβ-PIX rescued in vivo homing: 99.26% ±4.9 vs. 38.9% ±3.7 vs. 33.0%± 6.0; P< 0.0001 each mutant vs.Pak2WT/WT) proving the necessity of kinase activity and interaction with β-PIX for bone marrow homing. Taken together we show that both Pak2-kinase activity and its interaction with β-PIX exchange factor are required for coordinated HSPC F-actin formation and cell polarization, directed cell migration in vitro and homing to bone marrow in vivo. These data directly link the in vitro effects of Pak2 kinase with in vivo bonemarrow homing. Note All p values are calculated by Mann Whitney test. Disclosures: No relevant conflicts of interest to declare.

BackgroundPatients with RCC may achieve remission with immune-checkpoint inhibitors (ICI); however, most patients will progress. Adoptive cell therapy with autologous TIL allows for expansion of T-cells from tumor tissue leading to a polyclonal T-cell product with a diverse T-cell receptor repertoire capable of recognizing an array of tumor antigens. TIL therapy with centrally manufactured lifileucel demonstrated a 36% overall response rate in patients with ICI-refractory melanoma.1 We present our preclinical experience of TIL production in RCC.MethodsThis study was approved by the DF/HCC Institutional Review Board. Fresh tumor samples (≥1.5-cm) were harvested from consented patients undergoing resection for RCC. TIL were manufactured using pre-rapid expansion (1/10th scale) and rapid expansion (1/100th scale) protocol for 22 days. Characterization (total viable cells [TVC],% viability, identity, and potency) was performed on the final TIL product. TIL purity, differentiation, memory, activation, and exhaustion status were characterized using multi-color flow cytometry.ResultsBaseline characteristics of 11 recruited patients are shown in table 1. Clear cell was the most common histology (73%). Two patients had previously treated metastatic disease with samples harvested from the lung and adrenal gland; one patient had prior cryoablation with adjacent local recurrence. The remainder were treatment-naïve primary nephrectomy samples. Eight products (73%) showed acceptable TIL product attributes (table 2). Median (range) TVC, viability, and identity (CD45+CD3+%) for the final TIL product were 74×109 (18×109–133×109), 95% (86%–97%), and 98% (94%–99%), respectively. Median (range) percentage of CD4+ cells was 69% (21%–97%) and CD8+ cells was 27% (2%–72%). The non–T-cell population, including B cells, monocytes, and NK cells, was <7%. The final TIL product was functional, and responded to polyclonal bead stimulation; the median (range) IFNg and granzyme B were 8834 (3319–12,957) pg/mL and 34,329 (15,565–65,521) pg/mL, respectively. Acceptable TIL product was generated from both metastatic lesions and primary tumor samples. Of the 3 tumor samples that did not demonstrate acceptable TIL product attributes, one (ID 1) was from a patient treated with a CXCR4 inhibitor <1 month before resection, another (ID 4) was from a patient previously treated with ocrelizumab (CD20-directed cytolytic antibody) for multiple sclerosis, and the final (ID 8) was harvested from the cryoablated tumor nodule rather than the adjacent recurrent tumor.Abstract 176 Table 1Baseline demographics and tumor characteristicsAbstract 176 Table 2Summary of product attributes. 1, No CD3+ subset. 2, Product not available to testConclusionsThese feasibility data suggest that TIL can be successfully expanded ex vivo from RCC samples (including pre-treated and metastatic tumors) and support potential clinical investigation of TIL in patients with RCC.ReferencesSarnaik AA, Hamid O, Khushalani NI, Lewis KD, Medina T, Kluger HM, Thomas SS, Domingo-Musibay E, Pavlick AC, Whitman ED, Martin-Algarra S, Corrie P, Curti BD, Oláh J, Lutzky J, Weber JS, Larkin JMG, Shi W, Takamura T, Jagasia M, Qin H, Wu X, Chartier C, Graf Finckenstein F, Fardis M, Kirkwood JM, Chesney JA. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J Clin Oncol 2021 May 12:JCO2100612. doi: 10.1200/JCO.21.00612. Epub ahead of print. PMID: 33979178.Ethics ApprovalThis study was approved by the DF/HCC Institutional Review Board protocol # 06-105.

Abstract Background: Single-chain fragment variable region (scFv) in a chimeric antigen receptor (CAR) is a key component that directly binds the target antigen and transmits an activating signal into the CAR-T cells, subsequently triggering its effector function against the target cell. Thus, the affinity of scFv is considered to be critically important for CAR-T-cell function. However, optimal scFv affinity to induce maximal CAR function remains unclear. Methods: In this study, we constructed anti-CD20 scFv based on the reported sequence of humanized anti-CD20 monoclonal antibody with five different affinities: 20-vv (Kd value=7.07 nM), -fv (7.21), -fa (10.09), -sv (12.56), and -sa (14.66). Each scFv was then fused to an IgG4 hinge, a CD3-zeta chain, a CD28 costimulatory domain, and a truncated version of the epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD8-positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CARs on day 3, and enriched by selection with anti-EGFR mAb. CAR-T cells were expanded using anti-CD3/28 beads and used in the subsequent analysis. Results: To determine the lytic activity according to the scFv affinity, we tested cytotoxicity against CD20-transduced K562 cells (K562-CD20) by 51Cr releasing assay. CAR-T cells with the three highest-affinity scFv (20CAR-vv, -fv, -fa) effectively and equally lysed K562-CD20, while CAR-T cells with the two lowest-affinity scFv (20CAR-sv, -sa) failed to exhibit significant cytotoxicity (20CAR-vv, -fv, -fa, -sv, and -sa: 60.2±2.85%, 57.3±2.03%, 57.0±3.82%, 2.8±0.1%, and 2.0±0.64% at an E:T ratio = 10:1, respectively). To examine CAR-T-cell activation, cytokine secretion in the supernatant after 16 hours of culture with K562-CD20 was analyzed. Similarly to the cytotoxicity, 20CAR-vv, -fv, and -fa produced cytokine, but 20CAR-sv and -sa did not. The highest-affinity CAR-T (20CAR-vv) produced significantly higher IFN-g and IL-2 than the other CAR-T cells [IFN-g (pg/ml): 7644±326, 3380±179, 4718±315, 4.9±0.4, and 5.4±1.2; IL-2 (pg/ml): 4179±177, 1071±30, 1379±118, 0, and 0, respectively]. We next analyzed cell proliferation upon stimulation with K562-CD20 over 72 hours. 20CAR-vv resulted in rather poor absolute cell proliferation, while CAR-T cells with the second and third highest-affinity scFv (20CAR-fv, -fa) resulted in preferable proliferation (1.22±0.20-, 2.52±0.12-, 2.01±0.42-, 0.65±0.11-, and 1.15±0.13-fold expansion, respectively). To explore the possible reason for the difference of CAR-T proliferation, we evaluated activation-induced cell death (AICD) at 24 hours after the K562-CD20 stimulation (Annexin-V/PI staining). 20CAR-vv underwent significantly higher AICD than the other CAR-T cells (68.4±1.1, 39.6±0.7, 37.9±5.3, 25.9±11.2, and 16.1±1.2% cell death). Finally, we tested cell proliferation and AICD of the three highest-affinity scFv CAR-T cells (-vv, -fv, and -fa) upon CD20 high and low stimulation. In this experiment, we used CD20-transduced CEM cells, which are controlled to express high and low CD20: CEM-CD20 high (CD20 = 143,000 molecules/cell, the same level as other B-cell lines) and CEM-CD20 low (5,320 molecules/cell, the same level as the rituximab refractory cell line). The replacement of the stimulator from CD20-CEM high to low resulted in partial reduction of AICD to a level comparable to those of other 20CARs (-vv, 62.7%→33.0%, -fv, 56.0%→37.4%, –fa, 55.3%→27.7%), but the cell proliferation upon stimulation did not improve (-vv, 1.2-, -fv, 2.6-, -fa, 2.7-fold expansion after CD20-CEM low stim). These results suggest that the higher-affinity scFv was not necessarily associated with better CAR-T proliferation after ligation and may inhibit cell proliferation, possibly by multiple factors besides AICD. Conclusions: We observed that scFv with affinity above a certain level is needed to induce CAR-T function. Higher-affinity scFv induced more robust cytokine production; however, excessively high-affinity scFv in CAR can counteract T-cell proliferation, which may be partially associated with increased AICD. Optimization of scFv affinity is desirable for effective CAR-T production. Disclosures No relevant conflicts of interest to declare.

Cesium-137 has been worldwide used for estimation soil erosion. Its movement and transport in the environmental media depend on its retention by soil. Retention capacity of soil for radionuclides and pollutants as expressed by the distribution coefficient Kd (ratio of radionuclides retained by soil to those remained in liquid) that was estimated for 137Cs in some soils of Iraq. The soils were varied in clay 24.0-50.5%, organic mater 0.5-1.5%, and lime content 28.1-35.0%. Results showed that all the factors investigated were found to alter the values of Kd. The mean of Kd was 774 L kg-1 in the loam soil increased to 4375 L kg-1 in the clay soil when equilibrated with deionized water. These values were reduced to 402 and 1088 L kg-1 in chloride solution and 146 and 297 L kg-1 in groundwater for the loam and clay soils, respectively. The increased liquid to solid ratio showed a tendency to increase the distribution coefficient under the trace concentration of cesium. It was observed that an increase in the distribution coefficient of cesium from 1079 to 3312 L kg-1 when the liquid to solid ratio increased from 1:1 to 10:1. Differences in Kd were explained on the basis of ion exchange mechanism and indicate that certain test conditions. could be adopted by researchers for a precise estimate of the Kd. Such conditions should be corresponded as closely as possible to the natural field conditions.

Abstract Background Intravenous immunoglobulin (IVIG) resistance prediction is one of the primary clinical issues and study hotspots in KD. This study aimed to prospectively investigate the value of albumin-bilirubin grade (ALBI) in predicting IVIG resistance in KD and to assess whether ALBI has more predictive value or accuracy than either ALB or TBil alone in predicting IVIG resistance. Methods A total of 823 patients with KD were prospectively enrolled. The clinical and laboratory data were compared between the IVIG-response group (n = 708) and the IVIG-resistance group (n = 115). Multivariate logistic regression analysis was performed to identify the independent risk factors for IVIG resistance. Receiver operating characteristic (ROC) curves analysis was applied to assess the validity of ALBI, ALB, and TBil in predicting IVIG resistance. Results ALBI was significantly higher in patients with IVIG resistance and was identified as an independent risk factor for IVIG resistance in KD. The parameter of ALBI ≥ − 2.57 (AUC: 0.705, 95 %CI: 0.672–0.736), ALB ≤ 33.0 g/L (AUC: 0.659, 95 %CI: 0.626–0.692), and TBil ≥ 16.0µmol/L (AUC: 0.626, 95 %CI: 0.592–0.659), produced a sensitivity, specificity, PPV, and NPV of 0.617, 0.657, 0.226 and 0.914; 0.374, 0.850, 0.289 and 0.893; 0.269, 0.941, 0.425 and 0.888, respectively. Conclusions A higher ALBI was an independent risk factor for IVIG resistance in KD. It yielded better predictive ability than ALB and TBil alone for initial IVIG resistance.

Introduction: Kawasaki disease (KD), which is the most common multisystem vasculitis with unknown causes in childhood, causes coronary artery lesions (CALs). Treatment with a high dose of intravenous immunoglobulin (IVIG), plus steroids if needed, is the most effective therapy for the acute phase of KD. However, there are some very severe cases who need several times additional treatments and are at risk for CALs. In Japan, there are some scoring systems that initially predict IVIG-resistant patients. However, the problem is that these scoring systems fail in multiethnic populations. The aim of this study is to find universal biomarkers that predict treatment-resistant cases of KD. Methods: The subject was 276 KD patients, including Group 1 (n=214) who needed only 1 st line treatment, Group 2 (n=48) who needed 2 nd line treatment, Group 3 (n=14) who needed 3 rd line treatment or more. Tenascin C (TN-C), Pentraxin 3 (PTX3) and Procalcitonin (PCT) values, which were selected by systematic review, were measured before initial treatment in each group. Results: TN-C; 99.8±41.05 ng/ml in Group 1, 118.0±71.4 ng/ml in Group 2 and 183.0±25.0 ng/ml in Group 3. The TN-C level of Group 3 was significantly higher than that of all the others (p<0.01). The cutoff value for distinguishing Group 3 was 142.0 ng/ml (Area under the Curve (AUC)=0.81). PTX3; 16.2±9.0 ng/ml in Group 1, 31.4±19.7 ng/ml in Group 2 and 58.0±33.0 ng/ml in Group 3. The PTX3 level of Group 3 was significantly higher than that of all the others (p<0.01). The cutoff value for distinguishing Group 3 was 35.1 ng/ml (AUC=0.86). PCT; 0.79±0.77 ng/ml in Group 1, 2.55±3.01 ng/ml in Group 2 and 4.15±4.49 ng/ml in Group 3. The PCT level of Group 3 was significantly higher than that of all the others (p<0.01). The cutoff value for distinguishing Group 3 was 2.55 ng/ml (AUC=0.88). When those three biomarkers combined, Group 3 can be predicted with the sensitivity 79%, the specificity 96%, the positive predictive value 50% and the negative predictive value 99%. Conclusions: It may be possible to predict treatment-resistant KD cases with high sensitivity and specificity by combining the measurement from the universal biomarkers, TN-C, PTX3 and PCT, before initial treatment.

Abstract More than 48 kinase inhibitors (KIs) have been approved by Food and Drug Administration. However, drug-resistance (DR) eventually occurs, and secondary mutations have been found in the previously targeted primary-mutated cancer cells. Cancer and drug research communities recognize the importance of the kinase domain (KD) mutations for kinasopathies. So far, a systematic investigation of kinase mutations on DR hotspots has not been done yet. In this study, we systematically investigated four types of representative mutation hotspots (gatekeeper, G-loop, αC-helix and A-loop) associated with DR in 538 human protein kinases using large-scale cancer data sets (TCGA, ICGC, COSMIC and GDSC). Our results revealed 358 kinases harboring 3318 mutations that covered 702 drug resistance hotspot residues. Among them, 197 kinases had multiple genetic variants on each residue. We further computationally assessed and validated the epidermal growth factor receptor mutations on protein structure and drug-binding efficacy. This is the first study to provide a landscape view of DR-associated mutation hotspots in kinase’s secondary structures, and its knowledge will help the development of effective next-generation KIs for better precision medicine.

AbstractDNA polymerase ε (Polε) performs bulk synthesis of DNA on the leading strand during genome replication. Polε binds two substrates, a template:primer and dNTP, and catalyzes a covalent attachment of dNMP to the 3' end of the primer. Previous studies have shown that Polε easily inserts and extends ribonucleotides, which may promote mutagenesis and genome instability. In this work, we analyzed the mechanisms of discrimination against RNA-containing primers by human Polε (hPolε), performing binding and kinetic studies at near-physiological salt concentration. Pre-steady-state kinetic studies revealed that hPolεCD extends RNA primers with approximately 3300-fold lower efficiency in comparison to DNA, and addition of one dNMP to the 3′ end of an RNA primer increases activity 36-fold. Likewise, addition of one rNMP to the 3′ end of a DNA primer reduces activity 38-fold. The binding studies conducted in the presence of 0.15 M NaCl revealed that human hPolεCD has low affinity to DNA (KD of 1.5 µM). Strikingly, a change of salt concentration from 0.1 M to 0.15 M reduces the stability of the hPolεCD/DNA complex by 25-fold. Upon template:primer binding, the incoming dNTP and magnesium ions make hPolε discriminative against RNA and chimeric RNA–DNA primers. In summary, our studies revealed that hPolε discrimination against RNA-containing primers is based on the following factors: incoming dNTP, magnesium ions, a steric gate for the primer 2′OH, and the rigid template:primer binding pocket near the catalytic site. In addition, we showed the importance of conducting functional studies at near-physiological salt concentration.