Stephen E. Kurtz

Th17 Cytokines Stimulate CCL20 Expression in Keratinocytes In Vitro and In Vivo: Implications for Psoriasis Pathogenesis

T helper (Th) 17 cells have recently been implicated in psoriasis pathogenesis, but mechanisms of how these cells traffic into inflamed skin are unknown. By immunostaining for interleukin (IL)-17A and IL-22, we show numerous cells present in psoriasis lesions that produce these cytokines. We next found that Th17 cytokines (IL-17A, IL-22, and tumor necrosis factor (TNF)-alpha) markedly increased the expression of CC chemokine ligand (CCL) 20, a CC chemokine receptor (CCR)6 ligand, in human keratinocyte monolayer and raft cultures in a dose- and time-dependent manner. Lastly, we showed in mice that subcutaneous injection with recombinant IL-17A, IL-22, or TNF-alpha led to the upregulation of both CCL20 and CCR6 expression in skin as well as cutaneous T-cell infiltration. Taken together, these data show that Th17 cytokines stimulate CCL20 production in vitro and in vivo, and thus provide a potential explanation of how CCR6-positive Th17 cells maintain their continual presence in psoriasis through a positive chemotactic feedback loop.

IL-23 and IL-17A, but Not IL-12 and IL-22, Are Required for Optimal Skin Host Defense against Candida albicans

IL-23 and Th17 cells play important roles in host defense against systemic infections with extracellular bacteria and fungi, although their roles in immunity against localized skin infections are less well defined. Here, the contributions of IL-23 and Th17 cytokines in host defense against cutaneous Candida albicans infection were evaluated. Mice deficient in IL-23 or IL-17A demonstrated delayed healing and decreased IL-17A production after skin infection with C. albicans compared with wild-type mice or mice deficient in IL-12 or IL-22. Histologic examination revealed epidermal hyperplasia overlying infected dermis four days postinoculation in wild-type mice. In IL-23–deficient mice, fungal burden was greater in skin, neither IL-17A nor IL-22 mRNAs were expressed postinfection, and these mice demonstrated only minimal epidermal hyperplasia. Exogenous recombinant IL-17A injected at the site of skin infection promoted more rapid healing of candidiasis in both wild-type mice and mice deficient in IL-23 and IL-12. Taken together, these results demonstrate that IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal host defense against cutaneous candidiasis. In addition, recombinant IL-17A may serve as a potential therapy to enhance healing in individuals with chronic cutaneous candidiasis.

IL-23–Mediated Psoriasis-Like Epidermal Hyperplasia Is Dependent on IL-17A

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22−/− mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A−/− mice produced little ear swelling (p < 0.001, versus IL-23–injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23–induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23–induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23–mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.

Identification of a Peptide Derived from Vaccinia Virus A52R Protein That Inhibits Cytokine Secretion in Response to TLR-Dependent Signaling and Reduces In Vivo Bacterial-Induced Inflammation

TLRs recognize and respond to conserved motifs termed pathogen-associated molecular patterns. TLRs are characterized by an extracellular leucine-rich repeat motif and an intracellular Toll/IL-1R domain. Triggering of TLRs by pathogen-associated molecular patterns initiates a series of intracellular signaling events resulting in an inflammatory immune response designed to contain and eliminate the pathogen. Vaccinia virus encodes immunoregulatory proteins, such as A52R, that can effectively inhibit intracellular Toll/IL-1R signaling, resulting in a diminished host immune response and enhancing viral survival. In this study, we report the identification and characterization of a peptide derived from the A52R protein (sequence DIVKLTVYDCI) that, when linked to the nine-arginine cell transduction sequence, effectively inhibits cytokine secretion in response to TLR activation. The peptide had no effect on cytokine secretion resulting from cell activation that was initiated independent of TLR stimulation. Using a mouse model of otitis media with effusion, administration of heat-inactivated Streptococcus pneumoniae into the middle ears of BALB/c mice resulted in a significant inflammatory response that was dramatically reduced with peptide treatment. The identification of this peptide that selectively targets TLR-dependent signaling may have application in the treatment of chronic inflammation initiated by bacterial or viral infections.

Inflammatory Skin Disease in K5.hTGF-β1 Transgenic Mice Is Not Dependent on the IL-23/Th17 Inflammatory Pathway

In the presence of IL-6, transforming growth factor (TGF)-beta1 induces differentiation of T helper (Th) 17 cells in mice. Interleukin (IL)-23, a heterodimeric cytokine composed of IL-23p19 and IL-12/23p40 subunits, stimulates the growth and expansion of Th17 cells, and has been implicated in psoriasis pathogenesis. To study the associations between TGF-beta1, the IL-23/Th17 inflammatory pathway, and psoriasis, we investigated inflammatory skin disease in transgenic mice that constitutively overexpress human TGF-beta1 in basal keratinocytes (K5.hTGF-beta1 transgenic mice); these mice had previously been reported as having a psoriasis-like disease. K5.hTGF-beta1 transgenic mice had high levels of TGF-beta1 mRNA and protein in both skin and serum. Levels of cytokines involved in IL-23/Th17-mediated inflammation were not elevated in lesional skin compared with those in non-lesional and wild-type skin. It is noteworthy that IL-4 and IgE were markedly elevated in inflamed skin and serum, respectively, of transgenic mice. Monoclonal antibodies (mAbs) specifically directed against IL-23p19 or IL-12/23p40 had no clinical effect on established inflammatory skin disease in K5.hTGF-beta1 transgenic mice, whereas the same mAbs were able to block the development of murine experimental autoimmune encephalomyelitis, an IL-23/Th17-mediated disease. In summary, the IL-23/Th17 inflammatory pathway is not responsible for the maintenance of inflammatory skin disease in K5.hTGF-beta1 transgenic mice.

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies

Significance Mononuclear cells obtained from freshly isolated patient samples with various hematologic malignancies were evaluated for sensitivities to combinations of drugs that target specific cell-signaling pathways. The diagnostic, genetic/cytogenetic, and cellular features of the patient samples were correlated with effective drug combinations. For myeloid-derived tumors, such as acute myeloid leukemia, several combinations of targeted agents that include a kinase inhibitor and venetoclax, a selective inhibitor of BCL2, are effective. Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

Abstract DDT01-02: AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers

Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors. In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies. In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05). The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%). Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.

Functional genomic landscape of acute myeloid leukaemia.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML.Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

Inhibition of interleukin-1 receptor-associated kinase-1 is a therapeutic strategy for acute myeloid leukemia subtypes

Interleukin-1 receptor-associated kinase 1 (IRAK1), an essential mediator of innate immunity and inflammatory responses, is constitutively active in multiple cancers. We evaluated the role of IRAK1 in acute myeloid leukemia (AML) and assessed the inhibitory activity of multikinase inhibitor pacritinib on IRAK1 in AML. We demonstrated that IRAK1 is overexpressed in AML and provides a survival signal to AML cells. Genetic knockdown of IRAK1 in primary AML samples and xenograft model showed a significant reduction in leukemia burden. Kinase profiling indicated pacritinib has potent inhibitory activity against IRAK1. Computational modeling combined with site-directed mutagenesis demonstrated high-affinity binding of pacritinib to the IRAK1 kinase domain. Pacritinib exposure reduced IRAK1 phosphorylation in AML cells. A higher percentage of primary AML samples showed robust sensitivity to pacritinib, which inhibits FLT3, JAK2, and IRAK1, relative to FLT3 inhibitor quizartinib or JAK1/2 inhibitor ruxolitinib, demonstrating the importance of IRAK1 inhibition. Pacritinib inhibited the growth of AML cells harboring a variety of genetic abnormalities not limited to FLT3 and JAK2. Pacritinib treatment reduced AML progenitors in vitro and the leukemia burden in AML xenograft model. Overall, IRAK1 contributes to the survival of leukemic cells, and the suppression of IRAK1 may be beneficial among heterogeneous AML subtypes.