Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

Inflammatory networks underlying colorectal cancer.

Inflammation is emerging as one of the hallmarks of cancer, yet its role in most tumors remains unclear. Whereas a minority of solid tumors are associated with overt inflammation, long-term treatment with non-steroidal anti-inflammatory drugs is remarkably effective in reducing cancer rate and death. This indicates that inflammation might have many as-yet-unrecognized facets, among which an indolent course might be far more prevalent than previously appreciated. In this Review, we explore the various inflammatory processes underlying the development and progression of colorectal cancer and discuss anti-inflammatory means for its prevention and treatment.

Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.

Ectopic lymphoid-like structures (ELSs) are often observed in cancer, yet their function is obscure. Although ELSs signify good prognosis in certain malignancies, we found that hepatic ELSs indicated poor prognosis for hepatocellular carcinoma (HCC). We studied an HCC mouse model that displayed abundant ELSs and found that they constituted immunopathological microniches wherein malignant hepatocyte progenitor cells appeared and thrived in a complex cellular and cytokine milieu until gaining self-sufficiency. The egress of progenitor cells and tumor formation were associated with the autocrine production of cytokines previously provided by the niche. ELSs developed via cooperation between the innate immune system and adaptive immune system, an event facilitated by activation of the transcription factor NF-κB and abolished by depletion of T cells. Such aberrant immunological foci might represent new targets for cancer therapy.

The gut microbiome switches mutant p53 from tumour-suppressive to oncogenic.

Somatic mutations in p53, which inactivate the tumour-suppressor function of p53 and often confer oncogenic gain-of-function properties, are very common in cancer1,2. Here we studied the effects of hotspot gain-of-function mutations in Trp53 (the gene that encodes p53 in mice) in mouse models of WNT-driven intestinal cancer caused by Csnk1a1 deletion3,4 or ApcMin mutation5. Cancer in these models is known to be facilitated by loss of p533,6. We found that mutant versions of p53 had contrasting effects in different segments of the gut: in the distal gut, mutant p53 had the expected oncogenic effect; however, in the proximal gut and in tumour organoids it had a pronounced tumour-suppressive effect. In the tumour-suppressive mode, mutant p53 eliminated dysplasia and tumorigenesis in Csnk1a1-deficient and ApcMin/+ mice, and promoted normal growth and differentiation of tumour organoids derived from these mice. In these settings, mutant p53 was more effective than wild-type p53 at inhibiting tumour formation. Mechanistically, the tumour-suppressive effects of mutant p53 were driven by disruption of the WNT pathway, through preventing the binding of TCF4 to chromatin. Notably, this tumour-suppressive effect was completely abolished by the gut microbiome. Moreover, a single metabolite derived from the gut microbiota-gallic acid-could reproduce the entire effect of the microbiome. Supplementing gut-sterilized p53-mutant mice and p53-mutant organoids with gallic acid reinstated the TCF4-chromatin interaction and the hyperactivation of WNT, thus conferring a malignant phenotype to the organoids and throughout the gut. Our study demonstrates the substantial plasticity of a cancer mutation and highlights the role of the microenvironment in determining its functional outcome.

Immunity in acute myeloid leukemia: Where the immune response and targeted therapy meet.

Acute myeloid leukemia (AML) is a highly aggressive disease with high relapse and mortality rates. Recent years have shown a surge in novel therapeutic development for AML, both in clinical and preclinical stages. These developments include targeted therapies based on AML-specific molecular signatures as well as more general immune modulation and vaccination studies. In this review, we will explore the evolving arena of AML therapy and suggest some intriguing connections between immune system modulation and targeted therapy. Improved understanding of the immune system involvement in various stages of the disease and the crosstalk between immune effectors, targeted therapy, and AML cells can provide a better framework for designing the next generation of AML therapies.

Inflammation meets cancer, with NF-κB as the matchmaker.

Inflammation is a fundamental protective response that sometimes goes awry and becomes a major cofactor in the pathogenesis of many chronic human diseases, including cancer. Here we review the evolutionary relationship and opposing functions of the transcription factor NF-κB in inflammation and cancer. Although it seems to fulfill a distinctly tumor-promoting role in many types of cancer, NF-κB has a confounding role in certain tumors. Understanding the activity and function of NF-κB in the context of tumorigenesis is critical for its successful taming, an important challenge for modern cancer biology.

Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk.

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa-immune system crosstalk necessary for mounting protective T helper type 2 (T(H)2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1(Δgut)), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELMß, a key T(H)2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1(Δgut) mice with helminths favored a futile T(H)1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a T(H)2-facilitating epithelial cytokine; this indicated a T(H)2-amplification loop. We found that miR-375 was required for RELMß expression in vivo; miR-375-deficient mice had significantly less intestinal RELMß, which possibly explains the greater susceptibility of Dicer1(Δgut) mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.

Casein kinase 1-epsilon or 1-delta required for Wnt-mediated intestinal stem cell maintenance.

The intestinal epithelium holds an immense regenerative capacity mobilized by intestinal stem cells (ISCs), much of it supported by Wnt pathway activation. Several unique regulatory mechanisms ensuring optimal levels of Wnt signaling have been recognized in ISCs. Here, we identify another Wnt signaling amplifier, CKIε, which is specifically upregulated in ISCs and is essential for ISC maintenance, especially in the absence of its close isoform CKIδ. Co-ablation of CKIδ/ε in the mouse gut epithelium results in rapid ISC elimination, with subsequent growth arrest, crypt-villous shrinking, and rapid mouse death. Unexpectedly, Wnt activation is preserved in all CKIδ/ε-deficient enterocyte populations, with the exception of Lgr5+ ISCs, which exhibit Dvl2-dependent Wnt signaling attenuation. CKIδ/ε-depleted gut organoids cease proliferating and die rapidly, yet survive and resume self-renewal upon reconstitution of Dvl2 expression. Our study underscores a unique regulation mode of the Wnt pathway in ISCs, possibly providing new means of stem cell enrichment for regenerative medicine.

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

Casein kinase 1α (CK1α), a component of the ß-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by Csnk1a1) in skin physiology, we crossed mice harboring floxed Csnk1a1 with mice expressing K14-Cre-ERT2 to generate mice in which tamoxifen induces the deletion of Csnk1a1 exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by ß-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after Csnk1a1 ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ERT2 CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

ß-TrCP upregulates HIF-1 in prostate cancer cells.

The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (ß-TrCP) on the hypoxic pathway in cancer cells. ß-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that ß-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that ß-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of ß-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing ß-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.

mTOR generates an auto-amplification loop by triggering the ßTrCP- and CK1α-dependent degradation of DEPTOR.

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein ßTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the ßTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the ßTrCP-dependent degradation of DEPTOR via ßTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind ßTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and ßTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways.

Senescence-associated inflammatory responses: aging and cancer perspectives.

Senescent cells, albeit not proliferating, are metabolically and transcriptionally active, thereby capable of affecting their microenvironment, notably via the production of inflammatory mediators. These mediators maintain and propagate the senescence process to neighboring cells, and then recruit immune cells for clearing senescent cells. Among the inflammatory cues are molecules with pronounced tumor-controlling properties, both growth and invasion factors and inhibitory factors, working directly or via recruited immune cells. These senescence-inflammatory effects also prevail within tumors, mediated by the senescent tumor cells and the senescent tumor stroma. Here, we review the course and impact of senescence-associated inflammatory responses in aging and cancer. We propose that controlling senescence-associated inflammation by targeting specific inflammatory mediators may have a beneficial therapeutic effect in treatment of cancer and aging-related diseases.

Spermatogenesis rescue in a mouse deficient for the ubiquitin ligase SCF{beta}-TrCP by single substrate depletion.

beta-TrCP, the substrate recognition subunit of a Skp1-Cul1-F-box (SCF) ubiquitin ligase, is ubiquitously expressed from two distinct paralogs, targeting many regulatory proteins for proteasomal degradation. We generated inducible beta-TrCP hypomorphic mice and found that they are surprisingly healthy, yet have a severe testicular defect. We show that the two beta-TrCP paralogs have a nonredundant role in spermatogenesis. The testicular defect is tightly associated with cell adhesion failure within the seminiferous tubules and is fully reversible upon beta-TrCP restoration. Remarkably, testicular depletion of a single beta-TrCP substrate, Snail1, rescued the adhesion defect and restored spermatogenesis. Our studies highlight an unexpected functional reserve of this central E3, as well as a bottleneck in a specific tissue: a single substrate whose stabilization is incompatible with testicular differentiation.

CKIα ablation highlights a critical role for p53 in invasiveness control.

The mature gut renews continuously and rapidly throughout adult life, often in a damage-inflicting micro-environment. The major driving force for self-renewal of the intestinal epithelium is the Wnt-mediated signalling pathway, and Wnt signalling is frequently hyperactivated in colorectal cancer. Here we show that casein kinase Iα (CKIα), a component of the ß-catenin-destruction complex, is a critical regulator of the Wnt signalling pathway. Inducing the ablation of Csnk1a1 (the gene encoding CKIα) in the gut triggers massive Wnt activation, surprisingly without causing tumorigenesis. CKIα-deficient epithelium shows many of the features of human colorectal tumours in addition to Wnt activation, in particular the induction of the DNA damage response and cellular senescence, both of which are thought to provide a barrier against malignant transformation. The epithelial DNA damage response in mice is accompanied by substantial activation of p53, suggesting that the p53 pathway may counteract the pro-tumorigenic effects of Wnt hyperactivation. Notably, the transition from benign adenomas to invasive colorectal cancer in humans is typically linked to p53 inactivation, underscoring the importance of p53 as a safeguard against malignant progression; however, the mechanism of p53-mediated tumour suppression is unknown. We show that the maintenance of intestinal homeostasis in CKIα-deficient gut requires p53-mediated growth control, because the combined ablation of Csnk1a1 and either p53 or its target gene p21 (also known as Waf1, Cip1, Sdi1 and Cdkn1a) triggered high-grade dysplasia with extensive proliferation. Unexpectedly, these ablations also induced non-proliferating cells to invade the villous lamina propria rapidly, producing invasive carcinomas throughout the small bowel. Furthermore, in p53-deficient gut, loss of heterozygosity of the gene encoding CKIα caused a highly invasive carcinoma, indicating that CKIα functions as a tumour suppressor when p53 is inactivated. We identified a set of genes (the p53-suppressed invasiveness signature, PSIS) that is activated by the loss of both p53 and CKIα and which probably accounts for the brisk induction of invasiveness. PSIS transcription and tumour invasion were suppressed by p21, independently of cell cycle control. Restraining tissue invasion through suppressing PSIS expression is thus a novel tumour-suppressor function of wild-type p53.

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-ß.

In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or ß-catenin gene, activating the ß-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-ß/Smad functions. Most established CRC cell lines contain mutations in the TGF-ß/Smad pathway, but little is known about the function of TGF-ß in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-ß on the Lgr5(+) intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-ß-induced apoptosis in Apc-mutant organoids, including the Lgr5(+) stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-ß-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-ß than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-ß via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-ß-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-ß during intestinal adenoma development.

Critical role for IL-1ß in DNA damage-induced mucositis.

ß-TrCP, the substrate recognition subunit of SCF-type ubiquitin ligases, is ubiquitously expressed from two distinct paralogs, targeting for degradation many regulatory proteins, among which is the NF-κB inhibitor IκB. To appreciate tissue-specific roles of ß-TrCP, we studied the consequences of inducible ablation of three or all four alleles of the E3 in the mouse gut. The ablation resulted in mucositis, a destructive gut mucosal inflammation, which is a common complication of different cancer therapies and represents a major obstacle to successful chemoradiation therapy. We identified epithelial-derived IL-1ß as the culprit of mucositis onset, inducing mucosal barrier breach. Surprisingly, epithelial IL-1ß is induced by DNA damage via an NF-κB-independent mechanism. Tissue damage caused by gut barrier disruption is exacerbated in the absence of NF-κB, with failure to express the endogenous IL-1ß receptor antagonist IL-1Ra upon four-allele loss. Antibody neutralization of IL-1ß prevents epithelial tight junction dysfunction and alleviates mucositis in ß-TrCP-deficient mice. IL-1ß antagonists should thus be considered for prevention and treatment of severe morbidity associated with mucositis.

Acquisition of an immunosuppressive protumorigenic macrophage phenotype depending on c-Jun phosphorylation.

The inflamed tumor microenvironment plays a critical role in tumorigenesis. However, the mechanisms through which immune cells, particularly macrophages, promote tumorigenesis have only been partially elucidated, and the full scope of signaling pathways supplying macrophages with protumorigenic phenotypes still remain largely unknown. Here we report that germ-line absence of c-Jun N-terminal phosphorylation at serines 63 and 73 impedes inflammation-associated hepatocarcinogenesis, yet deleting c-Jun only in hepatocytes does not inhibit hepatocellular carcinoma (HCC) formation. Moreover, in human HCC-bearing livers, c-Jun phosphorylation is found in inflammatory cells, whereas it is mostly absent from malignant hepatocytes. Interestingly, macrophages in livers of mice with chronic hepatitis gradually switch their phenotype along the course of disease. Macrophage phenotype and density are dictated by c-Jun phosphorylation, in vitro and in vivo. Transition of macrophage phenotype, from antitumorigenic to protumorigenic, occurs before tumorigenesis, resulting in the production of various chemokines, including chemokine (C-C motif) ligand 17 (CCL17) and CCL22. Such signals, emanating from the liver microenvironment, direct the recruitment of regulatory T cells, which are known to facilitate HCC growth. Our findings identify c-Jun phosphorylation as a key mediator of macrophage education and point to the recruitment of immunosuppressive regulatory T cells as a possible protumorigenic mechanism.

Regulation of NF-κB by ubiquitination and degradation of the IκBs.

The nuclear factor-κB (NF-κB) signaling pathway is a busy ground for the action of the ubiquitin-proteasome system; many of the signaling steps are coordinated by protein ubiquitination. The end point of this pathway is to induce transcription, and to this end, there is a need to overcome a major obstacle, a set of inhibitors (IκBs) that bind NF-κB and prohibit either the nuclear entry or the DNA binding of the transcription factor. Two major signaling steps are required for the elimination of the inhibitors: activation of the IκB kinase (IKK) and degradation of the phosphorylated inhibitors. IKK activation and IκB degradation involve different ubiquitination modes; the latter is mediated by a specific E3 ubiquitin ligase SCF(ß-TrCP) . The F-box component of this E3, ß-TrCP, recognizes the IκB degron formed following phosphorylation by IKK and thus couples IκB phosphorylation to ubiquitination. SCF(ß-TrCP) -mediated IκB ubiquitination and degradation is a very efficient process, often resulting in complete degradation of the key inhibitor IκBα within a few minutes of cell stimulation. In vivo ablation of ß-TrCP results in accumulation of all the IκBs and complete NF-κB inhibition. As many details of IκB-ß-TrCP interaction have been worked out, the development of ß-TrCP inhibitors might be a feasible therapeutic approach for NF-κB-associated human disease. However, we may still need to advance our understanding of the mechanism of IκB degradation as well as of the diverse functions of ß-TrCP in vivo.

Metalloprotease type III effectors that specifically cleave JNK and NF-κB.

Two major arms of the inflammatory response are the NF-κB and c-Jun N-terminal kinase (JNK) pathways. Here, we show that enteropathogenic Escherichia coli (EPEC) employs the type III secretion system to target these two signalling arms by injecting host cells with two effector proteins, NleC and NleD. We provide evidence that NleC and NleD are Zn-dependent endopeptidases that specifically clip and inactivate RelA (p65) and JNK, respectively, thus blocking NF-κB and AP-1 activation. We show that NleC and NleD co-operate and complement other EPEC effectors in accomplishing maximal inhibition of IL-8 secretion. This is a remarkable example of a pathogen using multiple effectors to manipulate systematically the host inflammatory response signalling network.

Tissue-Predisposition to Cancer Driver Mutations.

Driver mutations are considered the cornerstone of cancer initiation. They are defined as mutations that convey a competitive fitness advantage, and hence, their mutation frequency in premalignant tissue is expected to exceed the basal mutation rate. In old terms, that translates to "the survival of the fittest" and implies that a selective process underlies the frequency of cancer driver mutations. In that sense, each tissue is its own niche that creates a molecular selective pressure that may favor the propagation of a mutation or not. At the heart of this stands one of the biggest riddles in cancer biology: the tissue-predisposition to cancer driver mutations. The frequency of cancer driver mutations among tissues is non-uniform: for instance, mutations in APC are particularly frequent in colorectal cancer, and 99% of chronic myeloid leukemia patients harbor the driver BCR-ABL1 fusion mutation, which is rarely found in solid tumors. Here, we provide a mechanistic framework that aims to explain how tissue-specific features, ranging from epigenetic underpinnings to the expression of viral transposable elements, establish a molecular basis for selecting cancer driver mutations in a tissue-specific manner.