Abstract Selective inhibition of the transcription elongation factor (P‐TEFb) complex represents a promising approach in cancer therapy, yet CDK9 inhibitors (CDK9i) are currently limited primarily to certain hematological malignancies. Herein, while initial responses to CDK9‐targeted therapies are observed in vitro across various KRAS‐mutant cancer types, their efficacy is far from satisfactory in nude mouse xenograft models. Mechanistically, CDK9 inhibition leads to compensatory activation of ERK‐MYC signaling, accompanied by the recovery of proto‐oncogenes, upregulation of immediate early genes (IEGs), stimulation of the complement C1r‐C3‐C3a cascade, and induction of tumor immunosuppression. The “paradoxical” regulation of PP2Ac activity involving the CDK9/Src interplay contributes to ERK phosphorylation and pause‐release of RNA polymerase II (Pol II). Co‐targeting of CDK9 and KRAS/MAPK signaling pathways eliminates ERK‐MYC activation and prevents feedback activation mediated by receptor tyrosine kinases, leading to more effective control of KRAS‐mutant cancers and overcoming KRASi resistance. Moreover, modulating the tumor microenvironment (TME) by complement system intervention enhances the response to CDK9i and potently suppresses tumor growth. Overall, the preclinical investigations establish a robust framework for conducting clinical trials employing KRASi/SOS1i/MEKi or immunomodifiers in combination with CDK9i to simultaneously target cancer cells and their crosstalk with the TME, thereby yielding improved responses in KRAS‐mutant patients.
Inhibitors targeting the antiapoptotic molecule BCL-2 have therapeutic potential for the treatment of acute myeloid leukaemia (AML); however, BCL-2 inhibitors such as venetoclax exhibit limited monotherapy efficacy in relapsed or refractory human AML. PI3Kδ/AKT signalling has been shown to be constitutively active in AML patients. Here, we demonstrate that the combination of BCL-2 and PI3Kδ inhibitors exerts synergistic antitumour effects both in vitro and in vivo in AML. Cotreatment with venetoclax and the specific PI3Kδ inhibitor idelalisib significantly enhanced antiproliferative effects and induced caspase-dependent apoptosis in a panel of AML cell lines. The synergistic effects were mechanistically based on the inactivation of AKT/4E-BP-1 signalling and the reduction of MCL-1 expression, which diminished the binding of Bim to MCL-1. Notably, compared with the parental FLT3-ITD-positive MV-4-11, the acquired FLT3 inhibitor quizartinib-resistant xenograft model carrying the F691L mutation, exhibited a markedly higher sensitivity to venetoclax. Furthermore, venetoclax combined with idelalisib led to tumour regression in all animals in this quizartinib-resistant AML model. Thus, these data indicate that combined inhibition of BCL-2 and PI3Kδ may be a promising strategy in AML, especially for patients with FLT3-ITD and/or FLT3-TKD mutations.
Despite initial success with FLT3 inhibitors (FLT3is), outcomes for FLT3-ITD acute myeloid leukemia (AML) patients remain unsatisfactory, underscoring the need for more effective treatment options. Epigenetic modifications, such as histone acetylation, contribute to AML's onset and persistence, advocating the potential for epigenetic therapies. However, the poor specificity of pan-histone deacetylase inhibitors (HDACis) leads to undesirable adverse effects, prompting the need for isoform-specific HDACis. This study aims to explore the antileukemic activities and mechanisms of IHCH9033, a novel class I HDACi, alone or combined with FLT3i in FLT3-ITD AML. The viability of AML cell lines and primary AML cells treated with HDACis alone or in combination with FLT3i was detected by MTT or CCK8 assay. Flow cytometry was utilized to examine cell apoptosis, cell cycle progression and ROS production. RNA sequencing analysis, RT-qPCR, western blotting, and co-immunoprecipitation assays were employed to elucidate the molecule mechanisms. The in vivo anti-leukemia efficacy was tested in xenografted mice models derived from FLT3-ITD cell lines and primary AML patients. Here, we identified IHCH9033, a novel selective class I HDACi, which exhibited an increased antitumor effect in FLT3-ITD AML through effectively eliminating leukemia burden and overcoming resistance to FLT3i. Mechanically, IHCH9033 selectively inhibited DNA repair in FLT3-ITD AML cells, leading to the accumulation of DNA damage that eventually resulted in cell cycle arrest and apoptosis. Additionally, IHCH9033 induced HSP90 acetylation, FLT3 ubiquitination, and proteasomal degradation of FLT3, thereby inhibiting FLT3 downstream signaling. Notably, IHCH9033 maintained its potency in both FLT3i-resistant AML cell lines and primary-resistant patient samples, and exerted strong synergy with the FLT3i quizartinib, leading to tumor regression in FLT3-ITD/TKD AML xenografts. In patient-derived xenografts, the treatment with IHCH9033, both alone and in combination, led to nearly complete eradication of the AML burden, without significant adverse effects. Our study shows that IHCH9033, a novel class I HDACi with a desirable pharmacological profile, is a promising drug candidate for FLT3-ITD AML, and suggests a strategy of combining class I HDACis and FLT3is in AML clinical trials to increase efficacy and overcome resistance, thus potentially providing a curative treatment option.
Abstract FMS-like tyrosine kinase-3 ( FLT3 ), a class 3 receptor tyrosine kinase, can be activated by mutations of internal tandem duplication (FLT3-ITD) or point mutations in the tyrosine kinase domain (FLT3-TKD), leading to constitutive activation of downstream signaling cascades, including the JAK/STAT5, PI3K/AKT/mTOR and RAS/MAPK pathways, which promote the progression of leukemic cells. Despite the initial promise of FLT3 inhibitors, the discouraging outcomes in the treatment of FLT3-ITD-positive acute myeloid leukemia (AML) promote the pursuit of more potent and enduring therapeutic approaches. The histone acetyltransferase complex comprising the E1A binding protein P300 and its paralog CREB-binding protein (p300/CBP) is a promising therapeutic target, but the development of effective p300/CBP inhibitors faces challenges due to inherent resistance and low efficacy, often exacerbated by the absence of reliable clinical biomarkers for patient stratification. In this study we investigated the role of p300/CBP in FLT3-ITD AML and evaluated the therapeutic potential of targeting p300/CBP alone or in combination with FLT3 inhibitors. We showed that high expression of p300 was significantly associated with poor prognosis in AML patients and positively correlated with FLT3 expression. We unveiled that the p300/CBP inhibitors A485 or CCS1477 dose-dependently downregulated FLT3 transcription via abrogation of histone acetylation in FLT3-ITD AML cells; in contrast, the FLT3 inhibitor quizartinib reduced the level of H3K27Ac. Concurrent inhibition of p300/CBP and FLT3 enhanced the suppression of FLT3 signaling and H3K27 acetylation, concomitantly reducing the phosphorylation of STAT5, AKT, ERK and the expression of c-Myc, thereby leading to synergistic antileukemic effects both in vitro and in vivo. Moreover, we found that p300/CBP-associated transcripts were highly expressed in quizartinib-resistant AML cells with FLT3-TKD mutation. Targeting p300/CBP with A485 or CCS1477 retained the efficacy of quizartinib, suggesting marked synergy when combined with p300/CBP inhibitors in quizartinib-resistant AML models, as well as primary FLT3-ITD + AML samples. These results demonstrate a potential therapeutic strategy of combining p300/CBP and FLT3 inhibitors to treat FLT3-ITD and FLT3-TKD AML.
KRASG12C inhibitors (KRASG12Ci) AMG510 and MRTX849 have shown promising efficacy in clinical trials and been approved for the treatment of KRASG12C-mutant cancers. However, the emergence of therapy-related drug resistance limits their long-term potential. This study aimed to identify the critical mediators and develop overcoming strategies.By using RNA sequencing, RT-qPCR and immunoblotting, we identified and validated the upregulation of c-Myc activity and the amplification of the long noncoding RNA ST8SIA6-AS1 in KRASG12Ci-resistant cells. The regulatory axis ST8SIA6-AS1/Polo-like kinase 1 (PLK1)/c-Myc was investigated by bioinformatics, RNA fluorescence in situ hybridization, RNA immunoprecipitation, RNA pull-down and chromatin immunoprecipitation. Gain/loss-of-function assays, cell viability assay, xenograft models, and IHC staining were conducted to evaluate the anti-cancer effects of co-inhibition of ST8SIA6-AS1/PLK1 pathway and KRAS both in vitro and in vivo.KRASG12Ci sustainably decreased c-Myc levels in responsive cell lines but not in cell lines with intrinsic or acquired resistance to KRASG12Ci. PLK1 activation contributed to this ERK-independent c-Myc stability, which in turn directly induced PLK1 transcription, forming a positive feedback loop and conferring resistance to KRASG12Ci. ST8SIA6-AS1 was found significantly upregulated in resistant cells and facilitated the proliferation of KRASG12C-mutant cancers. ST8SIA6-AS1 bound to Aurora kinase A (Aurora A)/PLK1 and promoted Aurora A-mediated PLK1 phosphorylation. Concurrent targeting of KRAS and ST8SIA6-AS1/PLK1 signaling suppressed both ERK-dependent and -independent c-Myc expression, synergistically led to cell death and tumor regression and overcame KRASG12Ci resistance.Our study deciphers that the axis of ST8SIA6-AS1/PLK1/c-Myc confers both intrinsic and acquired resistance to KRASG12Ci and represents a promising therapeutic target for combination strategies with KRASG12Ci in the treatment of KRASG12C-mutant cancers.
Histone deacetylase (HDAC) is an epigenetic antitumor drug target, but most existing HDAC inhibitors show limited antitumor activity and their use is often accompanied by serious adverse effects. To overcome these problems, we designed and synthesized a series of triazole-containing compounds as novel HDAC inhibitors. Among them, compound 19h exhibited potent and selective inhibition of HDAC1, with good antiproliferative activity in vitro and an excellent pharmacokinetic profile. Compound 19h significantly inhibited the growth of human tumor xenografts in nude mice and murine tumor growth in immune-competent mice bearing MC38 colon cancer. In the MC38 model, 19h increased the ratio of splenic CD4+ T effector cells and promoted complete tumor regression in 5/6 animals when combined with the mPD-1 antibody. These results suggested that selective class I HDAC inhibitors exert direct tumor growth inhibition and indirect immune cell-mediated antitumor effects and are synergistic with immune checkpoint inhibitors.
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