SIBP-03, a novel anti-HER3 antibody, exerts antitumor effects and synergizes with EGFR- and HER2-targeted drugs.

HER3 (human epidermal growth factor receptor 3) acts through heterodimerization with EGFR (epidermal growth factor receptor) or HER2 to play an essential role in activating phosphoinositide 3-kinase (PI3K) and AKT signaling-a crucial pathway that promotes tumor cell survival. HER3 is a promising target for cancer therapy, and several HER3-directed antibodies have already entered into clinical trials. In this study we characterized a novel anti-HER3 monoclonal antibody, SIBP-03. SIBP-03 (0.01-10 µg/mL) specifically and concentration-dependently blocked both neuregulin (NRG)-dependent and -independent HER3 activation, attenuated HER3-mediated downstream signaling and inhibited cell proliferation. This antitumor activity was dependent, at least in part, on SIBP-03-induced, cell-mediated cytotoxicity and cellular phagocytosis. Importantly, SIBP-03 enhanced the antitumor activity of EGFR- or HER2-targeted drugs (cetuximab or trastuzumab) in vitro and in vivo. The mechanisms underlying this synergy involve increased inhibition of HER3-mediated downstream signaling. Collectively, these results demonstrated that SIBP-03, which is currently undergoing a Phase I clinical trial in China, may offer a new treatment option for patients with cancers harboring activated HER3, particularly as part of a combinational therapeutic strategy.

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy, exerts antitumor activity against colon cancer.

Hsp90 is a potential therapeutic target for tumor, as it maintains the stability of a variety of proteins related to tumor development and progression. Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth. In this study, we identified DCZ5248, a triazine derivative, was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo. We showed that DCZ5248 (0.1-10 µM) induced dose-dependent degradation of Hsp90 client proteins (AKT, CDK4, CDK6 and RAF-1) in HCT 116 colon cancer cells through a proteasome-dependent pathway. Meanwhile, DCZ5248 (0.3 µM) induced cytoplasmic vacuole formation, LC3 II conversion, p62 protein upregulation, and inhibited autophagy at the late stage in the colon cancer cell lines tested. We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity. The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown. Importantly, inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest, apoptosis, and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo. These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.

Pd-Catalyzed Decarboxylative Olefination: Stereoselective Synthesis of Polysubstituted Butadienes and Macrocyclic P-glycoprotein Inhibitors.

The efficient and stereoselective synthesis of polysubstituted butadienes, especially the multifunctional butadienes, represents a great challenge in organic synthesis. Herein, we wish to report a distinctive Pd(0) carbene-initiated decarboxylative olefination approach that enables the direct coupling of diazo esters with vinylethylene carbonates (VECs), vinyl oxazolidinones, or vinyl benzoxazinones to afford alcohol-, amine-, or aniline-containing 1,3-dienes in moderate to high yields and with excellent stereoselectivity. This protocol features operational simplicity, mild reaction conditions, a broad substrate scope, and gram-scalability. Notably, a structurally unique allylic Pd(II) intermediate was isolated and characterized. DFT calculation and control experiments demonstrated that a rare Pd(0) carbene intermediate could be involved in this reaction. Moreover, the polysubstituted butadienes as novel building blocks were unprecedentedly assembled into macrocycles, which efficiently inhibited the P-glycoprotein and dramatically reversed multidrug resistance in cancer cells by 190-fold.

YES1 amplification confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive cancer.

BACKGROUND: Trastuzumab-emtansine (T-DM1), one of the most potent HER2-targeted drugs, shows impressive efficacy in patients with HER2-positive breast cancers. However, resistance inevitably occurs and becomes a critical clinical problem. METHODS: We modelled the development of acquired resistance by exposing HER2-positive cells to escalating concentrations of T-DM1. Signalling pathways activation was detected by western blotting, gene expression was analysed by qRT-PCR and gene copy numbers were determined by qPCR. The role of Yes on resistance was confirmed by siRNA-mediated knockdown and stable transfection-mediated overexpression. The in vivo effects were tested in xenograft model. RESULTS: We found that Yes is overexpressed in T-DM1-resistant cells owing to amplification of chromosome region 18p11.32, where the YES1 gene resides. Yes activated multiple proliferation-related signalling pathways, including EGFR, PI3K and MAPK, and led to cross-resistance to all types of HER2-targeted drugs, including antibody-drug conjugate, antibody and small molecule inhibitor. The outcome of this cross-resistance may be a clinically incurable condition. Importantly, we found that inhibiting Yes with dasatinib sensitised resistant cells in vitro and in vivo. CONCLUSIONS: Our study revealed that YES1 amplification conferred resistance to HER2-targeted drugs and suggested the potential application of the strategy of combining HER2 and Yes inhibition in the clinic.

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

Pharmacological characterization of TQ05310, a potent inhibitor of isocitrate dehydrogenase 2 R140Q and R172K mutants.

Isocitrate dehydrogenase 2 (IDH2), an important mitochondrial metabolic enzyme involved in the tricarboxylic acid cycle, is mutated in a variety of cancers. AG-221, an inhibitor primarily targeting the IDH2-R140Q mutant, has shown remarkable clinical benefits in the treatment of relapsed or refractory acute myeloid leukemia patients. However, AG-221 has weak inhibitory activity toward IDH2-R172K, a mutant form of IDH2 with more severe clinical manifestations. Herein, we report TQ05310 as the first mutant IDH2 inhibitor that potently targets both IDH2-R140Q and IDH2-R172K mutants. TQ05310 inhibited mutant IDH2 enzymatic activity, suppressed (R)-2-hydroxyglutarate (2-HG) production and induced differentiation in cells expressing IDH2-R140Q and IDH2-R172K, but not in cells expressing wild-type IDH1/2 or mutant IDH1. TQ05310 bound to both IDH2-R140Q and IDH2-R172K, with Q316 being the critical residue mediating the binding of TQ05310 with IDH2-R140Q, but not with IDH2-R172K. TQ05310 also had favorable pharmacokinetic characteristics and profoundly inhibited 2-HG production in a tumor xenografts model. The results of the current study establish a solid foundation for further clinical investigation of TQ05310, and provide new insight into the development of novel mutant IDH2 inhibitors.

SHR-A1403, a novel c-mesenchymal-epithelial transition factor (c-Met) antibody-drug conjugate, overcomes AZD9291 resistance in non-small cell lung cancer cells overexpressing c-Met.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have been used as the first-line treatment of non-small cell lung cancers (NSCLC) harboring EGFR-activating mutations, but acquired resistance is ubiquitous and needs to be solved urgently. Here, we introduce an effective approach for overcoming resistance to the EGFR-TKI, AZD9291, in NSCLC cells using SHR-A1403, a novel c-mesenchymal-epithelial transition factor (c-Met)-targeting antibody-drug conjugate (ADC) consisting of an anti-c-Met monoclonal antibody (c-Met mAb) conjugated to a microtubule inhibitor. Resistant cells were established by exposing HCC827 to increasing concentrations of EGFR-TKI. c-Met was found to be overexpressed in most resistant cells. AZD9291 resistance was partially restored by combination of AZD9291 and crizotinib only in resistant cells overexpressing phospho-c-Met, which synergistically inhibited c-Met-mediated phosphorylation of the downstream targets ERK1/2 and AKT. In resistant cells overexpressing c-Met, neither crizotinib nor c-Met mAb was able to overcome AZD9291 resistance. In contrast, SHR-A1403 strongly inhibited proliferation of AZD9291-resistant HCC827 overexpressing c-Met, regardless of the levels of c-Met phosphorylation. SHR-A1403 bound to resistant cells overexpressing c-Met was internalized into cells and released associated microtubule inhibitor, resulting in cell-killing activity that was dependent on c-Met expression levels only, irrespective of the involvement of c-Met or EGFR signaling in AZD9291 resistance. Consistent with its activity in vitro, SHR-A1403 significantly inhibited the growth of AZD9291-resistant HCC827 tumors and caused tumor regression in vivo. Thus, our findings show that SHR-A1403 efficiently overcomes AZD9291 resistance in cells overexpressing c-Met, and further indicate that c-Met expression level is a biomarker predictive of SHR-A1403 efficacy.

Preclinical characterization of SHR6390, a novel CDK 4/6 inhibitor, in vitro and in human tumor xenograft models.

Inhibition of the cyclin-dependent kinase (CDK) 4/6-retinoblastoma (RB) pathway is an effective therapeutic strategy against cancer. Here, we performed a preclinical investigation of the antitumor activity of SHR6390, a novel CDK4/6 inhibitor. SHR6390 exhibited potent antiproliferative activity against a wide range of human RB-positive tumor cells in vitro, and exclusively induced G1 arrest as well as cellular senescence, with a concomitant reduction in the levels of Ser780-phosphorylated RB protein. Compared with the well-known CDK4/6 inhibitor palbociclib, orally administered SHR6390 led to equivalent or improved tumor efficacy against a panel of carcinoma xenografts, and produced marked tumor regression in some models, in association with sustained target inhibition in tumor tissues. Furthermore, SHR6390 overcame resistance to endocrine therapy and HER2-targeting antibody in ER-positive and HER2-positive breast cancer, respectively. Moreover, SHR6390 combined with endocrine therapy exerted remarkable synergistic antitumor activity in ER-positive breast cancer. Taken together, our findings indicate that SHR6390 is a novel CDK4/6 inhibitor with favorable pharmaceutical properties for use as an anticancer agent.

Pharmacologic characterization of fluzoparib, a novel poly(ADP-ribose) polymerase inhibitor undergoing clinical trials.

Poly(ADP-ribose) polymerase (PARP) enzymes play an important role in repairing DNA damage and maintaining genomic stability. Olaparib, the first-in-class PARP inhibitor, has shown remarkable clinical benefits in the treatment of BRCA-mutated ovarian or breast cancer. However, the undesirable hematological toxicity and pharmacokinetic properties of olaparib limit its clinical application. Here, we report the first preclinical characterization of fluzoparib (code name: SHR-3162), a novel, potent, and orally available inhibitor of PARP. Fluzoparib potently inhibited PARP1 enzyme activity and induced DNA double-strand breaks, G2 /M arrest, and apoptosis in homologous recombination repair (HR)-deficient cells. Fluzoparib preferentially inhibited the proliferation of HR-deficient cells and sensitized both HR-deficient and HR-proficient cells to cytotoxic drugs. Notably, fluzoparib showed good pharmacokinetic properties, favorable toxicity profile, and superior antitumor activity in HR-deficient xenografts models. Furthermore, fluzoparib in combination with apatinib or with apatinib plus paclitaxel elicited significantly improved antitumor responses without extra toxicity. Based on these findings, studies to evaluate the efficacy and safety of fluzoparib (phase II) and those two combinations (phase I) have been initiated. Taken together, our results implicate fluzoparib as a novel attractive PARP inhibitor.

SHR-A1403, a novel c-Met antibody-drug conjugate, exerts encouraging anti-tumor activity in c-Met-overexpressing models.

Emerging evidence demonstrates that a c-Met antibody-drug conjugate (ADC) has superior efficacy and safety profiles compared with those of currently available small molecules or antibody inhibitors for the treatment of c-Met-overexpressing cancers. Here we described both the in vitro and in vivo efficacies of SHR-A1403, a novel c-Met ADC composed of a humanized IgG2 monoclonal antibody against c-Met conjugated to a novel cytotoxic microtubule inhibitor. SHR-A1403 showed high affinity to c-Met proteins derived from human or monkey and potent inhibitory effects in cancer cell lines with high c-Met protein expression. In mice bearing tumors derived from cancer cell lines or patient HCC tissues with confirmed c-Met overexpression, SHR-A1403 showed excellent anti-tumor efficacy. Antibody binding with c-Met contributed to SHR-A1403 endocytosis; the subsequent translocation to lysosomes and cytotoxicity of the released toxin are speculated to be predominant mechanisms underlying the anti-tumor activity of SHR-A1403. In conclusion, SHR-A1403 showed significant anti-tumor activity in cancer cell lines, xenograft mouse models and an HCC PDX model, which all have high c-Met levels. These data provide references for SHR-A1403 as a potential therapy for the treatment of cancers with c-Met overexpression.

Aberrant modulation of ribosomal protein S6 phosphorylation confers acquired resistance to MAPK pathway inhibitors in BRAF-mutant melanoma.

BRAF and MEK inhibitors have shown remarkable clinical efficacy in BRAF-mutant melanoma; however, most patients develop resistance, which limits the clinical benefit of these agents. In this study, we found that the human melanoma cell clones, A375-DR and A375-TR, with acquired resistance to BRAF inhibitor dabrafenib and MEK inhibitor trametinib, were cross resistant to other MAPK pathway inhibitors. In these resistant cells, phosphorylation of ribosomal protein S6 (rpS6) but not phosphorylation of ERK or p90 ribosomal S6 kinase (RSK) were unable to be inhibited by MAPK pathway inhibitors. Notably, knockdown of rpS6 in these cells effectively downregulated G1 phase-related proteins, including RB, cyclin D1, and CDK6, induced cell cycle arrest, and inhibited proliferation, suggesting that aberrant modulation of rpS6 phosphorylation contributed to the acquired resistance. Interestingly, RSK inhibitor had little effect on rpS6 phosphorylation and cell proliferation in resistant cells, whereas P70S6K inhibitor showed stronger inhibitory effects on rpS6 phosphorylation and cell proliferation in resistant cells than in parental cells. Thus regulation of rpS6 phosphorylation, which is predominantly mediated by BRAF/MEK/ERK/RSK signaling in parental cells, was switched to mTOR/P70S6K signaling in resistant cells. Furthermore, mTOR inhibitors alone overcame acquired resistance and rescued the sensitivity of the resistant cells when combined with BRAF/MEK inhibitors. Taken together, our findings indicate that RSK-independent phosphorylation of rpS6 confers resistance to MAPK pathway inhibitors in BRAF-mutant melanoma, and that mTOR inhibitor-based regimens may provide alternative strategies to overcome this acquired resistance.

Synthesis and antitumor activity of a series of lactone-opened camptothecin derivatives.

A series of E-ring lactone-opened camptothecin (CPT) derivatives bearing with terminal aza-heterocyclic groups were synthesized, and their antitumor activity was evaluated both in vitro and in vivo. Hydroxyl-amide analogues with morpholin-4-yl displayed excellent antitumor activity in vitro and efficient inhibition on tumor xenograph model in nude mice. Ester-amide compounds acted less active in vitro cytotoxicity and lower inhibition activity in vivo. Substitutions at 7- and 10- positions favored the antitumor activity.

Pharmacological characterization of hetrombopag, a novel orally active human thrombopoietin receptor agonist.

Nonpeptide thrombopoietin receptor (TPOR/MPL) agonists, such as eltrombopag, have been used to treat thrombocytopenia of various aetiologies. Here, we investigated the pharmacological properties of hetrombopag, a new orally active small-molecule TPOR agonist, in preclinical models. Hetrombopag specifically stimulated proliferation and/or differentiation of human TPOR-expressing cells, including 32D-MPL and human hematopoietic stem cells, with low nanomolar EC50 values through stimulation of STAT, PI3K and ERK signalling pathways. Notably, hetrombopag effectively up-regulated G1 -phase-related proteins, including p-RB, Cyclin D1 and CDK4/6, normalized progression of the cell cycle, and prevented apoptosis by modulating BCL-XL/BAK expression in 32D-MPL cells. Moreover, hetrombopag and TPO acted additively in stimulating TPOR-dependent signalling, promoting cell viability, and preventing apoptosis. Orally administered hetrombopag specifically promoted the viability and growth of 32D-MPL cells in hollow fibres implanted into nude mice with much higher potency than that of the well-known TPOR agonist, eltrombopag, in association with activation of TPOR-dependent signal transduction in vivo. Taken together, our findings indicate that, given its favourable pharmacological characteristics, hetrombopag may represent a new, orally active, small-molecule TPOR agonist for patients with thrombocytopenia.

Preclinical characterization of anlotinib, a highly potent and selective vascular endothelial growth factor receptor-2 inhibitor.

Abrogating tumor angiogenesis by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2) has been established as a therapeutic strategy for treating cancer. However, because of their low selectivity, most small molecule inhibitors of VEGFR2 tyrosine kinase show unexpected adverse effects and limited anticancer efficacy. In the present study, we detailed the pharmacological properties of anlotinib, a highly potent and selective VEGFR2 inhibitor, in preclinical models. Anlotinib occupied the ATP-binding pocket of VEGFR2 tyrosine kinase and showed high selectivity and inhibitory potency (IC50 <1 nmol/L) for VEGFR2 relative to other tyrosine kinases. Concordant with this activity, anlotinib inhibited VEGF-induced signaling and cell proliferation in HUVEC with picomolar IC50 values. However, micromolar concentrations of anlotinib were required to inhibit tumor cell proliferation directly in vitro. Anlotinib significantly inhibited HUVEC migration and tube formation; it also inhibited microvessel growth from explants of rat aorta in vitro and decreased vascular density in tumor tissue in vivo. Compared with the well-known tyrosine kinase inhibitor sunitinib, once-daily oral dose of anlotinib showed broader and stronger in vivo antitumor efficacy and, in some models, caused tumor regression in nude mice. Collectively, these results indicate that anlotinib is a well-tolerated, orally active VEGFR2 inhibitor that targets angiogenesis in tumor growth, and support ongoing clinical evaluation of anlotinib for a variety of malignancies.

STAT3 activation confers trastuzumab-emtansine (T-DM1) resistance in HER2-positive breast cancer.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate that has been approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. Despite the remarkable efficacy of T-DM1 in many patients, resistance to this therapeutic has emerged as a significant clinical problem. In the current study, we used BT-474/KR cells, a T-DM1-resistant cell line established from HER2-positive BT-474 breast cancer cells, as a model to investigate mechanisms of T-DM1 resistance and explore effective therapeutic regimens. We show here for the first time that activation of signal transducer and activator of transcription 3 (STAT3) mediated by leukemia inhibitory factor receptor (LIFR) overexpression confers resistance to T-DM1. Moreover, secreted factors induced by activated STAT3 in resistant cells limit the responsiveness of cells that were originally sensitive to T-DM1. Importantly, STAT3 inhibition sensitizes resistant cells to T-DM1, both in vitro and in vivo, suggesting that the combination T-DM1 with STAT3-targeted therapy is a potential treatment for T-DM1-refractory patients.

Pharmacokinetics and disposition of anlotinib, an oral tyrosine kinase inhibitor, in experimental animal species.

Anlotinib is a new oral tyrosine kinase inhibitor; this study was designed to characterize its pharmacokinetics and disposition. Anlotinib was evaluated in rats, tumor-bearing mice, and dogs and also assessed in vitro to characterize its pharmacokinetics and disposition and drug interaction potential. Samples were analyzed by liquid chromatography/mass spectrometry. Anlotinib, having good membrane permeability, was rapidly absorbed with oral bioavailability of 28%-58% in rats and 41%-77% in dogs. Terminal half-life of anlotinib in dogs (22.8±11.0 h) was longer than that in rats (5.1±1.6 h). This difference appeared to be mainly associated with an interspecies difference in total plasma clearance (rats, 5.35±1.31 L·h-1·kg-1; dogs, 0.40±0.06 L·h-1/kg-1). Cytochrome P450-mediated metabolism was probably the major elimination pathway. Human CYP3A had the greatest metabolic capability with other human P450s playing minor roles. Anlotinib exhibited large apparent volumes of distribution in rats (27.6±3.1 L/kg) and dogs (6.6±2.5 L/kg) and was highly bound in rat (97%), dog (96%), and human plasma (93%). In human plasma, anlotinib was predominantly bound to albumin and lipoproteins, rather than to α1-acid glycoprotein or γ-globulins. Concentrations of anlotinib in various tissue homogenates of rat and in those of tumor-bearing mouse were significantly higher than the associated plasma concentrations. Anlotinib exhibited limited in vitro potency to inhibit many human P450s, UDP-glucuronosyltransferases, and transporters, except for CYP3A4 and CYP2C9 (in vitro half maximum inhibitory concentrations, <1 µmol/L). Based on early reported human pharmacokinetics, drug interaction indices were 0.16 for CYP3A4 and 0.02 for CYP2C9, suggesting that anlotinib had a low propensity to precipitate drug interactions on these enzymes. Anlotinib exhibits many pharmacokinetic characteristics similar to other tyrosine kinase inhibitors, except for terminal half-life, interactions with drug metabolizing enzymes and transporters, and plasma protein binding.

Aberrant intracellular metabolism of T-DM1 confers T-DM1 resistance in human epidermal growth factor receptor 2-positive gastric cancer cells.

Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) consisting of human epidermal growth factor receptor 2 (HER2)-targeted mAb trastuzumab linked to antimicrotubule agent mertansine (DM1), has been approved for the treatment of HER2-positive metastatic breast cancer. Acquired resistance has been a major obstacle to T-DM1 treatment, and mechanisms remain incompletely understood. In the present study, we established a T-DM1-resistant N87-KR cell line from HER2-positive N87 gastric cancer cells to investigate mechanisms of acquired resistance and develop strategies for overcoming it. Although the kinetics of binding, internalization, and externalization of T-DM1 were the same in N87-KR cells and N87 cells, N87-KR was strongly resistant to T-DM1, but remained sensitive to both trastuzumab and DM1. T-DM1 failed to inhibit microtubule polymerization in N87-KR cells. Consistently, lysine-MCC-DM1, the active T-DM1 metabolite that inhibits microtubule polymerization, accumulated much less in N87-KR cells than in N87 cells. Furthermore, lysosome acidification, achieved by vacuolar H+ -ATPase (V-ATPase), was much diminished in N87-KR cells. Notably, treatment of sensitive N87 cells with the V-ATPase selective inhibitor bafilomycin A1 induced T-DM1 resistance, suggesting that aberrant V-ATPase activity decreases T-DM1 metabolism, leading to T-DM1 resistance in N87-KR cells. Interestingly, HER2-targeted ADCs containing a protease-cleavable linker, such as hertuzumab-vc-monomethyl auristatin E, were capable of efficiently overcoming this resistance. Our results show for the first time that a decrease in T-DM1 metabolites induced by aberrant V-ATPase activity contributes to T-DM1 resistance, which could be overcome by HER2-targeted ADCs containing different linkers, including a protease-cleavable linker. Accordingly, we propose that V-ATPase activity in lysosomes is a novel biomarker for predicting T-DM1 resistance.

Puquitinib, a novel orally available PI3Kδ inhibitor, exhibits potent antitumor efficacy against acute myeloid leukemia.

The PI3Kδ isoform (PIK3CD), also known as P110δ, is predominately expressed in leukocytes and has been implicated as a potential target in the treatment of hematological malignancies. In this report, we detailed the pharmacologic properties of puquitinib, a novel, orally available PI3Kδ inhibitor. Puquitinib, which binds to the ATP-binding pocket of PI3Kδ, was highly selective and potent for PI3Kδ relative to other PI3K isoforms and a panel of protein kinases, exhibiting low-nanomolar biochemical and cellular inhibitory potencies. Additional cellular profiling demonstrated that puquitinib inhibited proliferation, induced G1 -phase cell-cycle arrest and apoptosis in acute myeloid leukemia (AML) cell lines, through downregulation of PI3K signaling. In in vivo AML xenografts, puquitinib alone showed stronger efficacy than the well-known p110δ inhibitor, CAL-101, in association with a reduction in AKT and ERK phosphorylation in tumor tissues, without causing noticeable toxicity. Furthermore, the combination of puquitinib with cytotoxic drugs, especially daunorubicin, yielded significantly stronger antitumor efficacy compared with each agent alone. Thus, puquitinib is a promising agent with pharmacologic properties that are favorable for the treatment of AML.

AKT is critically involved in the antagonism of BRAF inhibitor sorafenib against dabrafenib in colorectal cancer cells harboring both wild-type and mutant (V600E) BRAF genes.

BRAF, one of the key factors in mitogen-activated protein kinase (MAPK) signaling pathway, plays an important role in cell functions including growth and proliferation. Inhibition of BRAF represents a promising antitumor strategy. Dabrafenib, a type I inhibitor of BRAF interrupting RAF/MEK interaction, has been approved by FDA as a single agent or combined with MEK inhibitor trametinib for the treatment of patients with BRAF V600E mutation-positive advanced melanoma. In the present study, we investigated the feasibility of combined treatment with dabrafenib and sorafenib, type I and type II BRAF inhibitor respectively, on colorectal cancer cells with BRAF V600E mutation. Unexpectedly, sorafenib significantly antagonized the inhibition effect of dabrafenib on the proliferation of colorectal cancer HT-29 and Colo205 cells. The antagonism relied on co-existence of wild-type and mutant (V600E) BRAF, for no antagonism was observed in tumor cells expressing homozygous wild-type or mutant (V600E) BRAF. BRAF, but not CRAF, was required for this antagonism. Moreover, we found that sorafenib reversed dabrafenib inhibition of AKT in HT-29 cells, and phosphatidylinositol-3-kinase (PI3K) inhibitor GDC0941 significantly restored this antagonistic effect when combined with dabrafenib and sorafenib, indicating that AKT is critically involved in this antagonism. Collectively, we found that significant antagonism was observed when dabrafenib was combined with sorafenib in colorectal cancer cells harboring heterozygous genotype of BRAF and AKT is critically involved in this antagonism. We suggest that BRAF inhibitor dabrafenib and sorafenib should not be combined in clinic.