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Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and l-amino acids ([D]-K6L9) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, ∼10.5) for histidines (pKa, ∼6.1) in the parental peptide [D]-K6L9. Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K3H3L9 and [D]-H6L9. Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H6L9 mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors. [Cancer Res 2009;69(8):3458–63]

Supplementary Figures 1-3 from Suppression of Human Solid Tumor Growth in Mice by Intratumor and Systemic Inoculation of Histidine-Rich and pH-Dependent Host Defense–like Lytic Peptides

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.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C + monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.

Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Type I interferons (IFNα, IFNβ) are key regulators of innate and adaptive immunity, modulating the severity of both viral and bacterial infections. While type I IFN signaling leads to improved outcomes in viral infections, its role in bacterial infections is more contextual and depends on the specific pathogen and route of infection. Given the limited evidence on whether type I IFN signaling affects enteric bacterial pathogens, we investigated the role of this signaling pathway in Salmonella enterica serovar Typhimurium (S. typhimurium)-induced colitis. Comparing mice deficient in IFNAR1- the common receptor for IFNα and IFNβ- with wild-type mice, we found that type I IFN signaling leads to more rapid death, more severe colonic inflammation, higher serum levels of pro-inflammatory cytokines, and greater bacterial dissemination. Specific ablation of plasmacytoid dendritic cells (pDCs), which are prominent producers of type I IFNs in antiviral responses, did not alter survival after infection. This result established that pDCs do not play a major role in the pathogenesis of S. typhimurium colitis. Flow cytometric analysis of macrophages and conventional dendritic cells (cDCs) during active colitis demonstrated an increase in CD11c- macrophages and CD103+ cDCs in the colon of Ifnar1-/- animals. Interestingly, cells expressing the anti-inflammatory cytokine receptor IL-10R are more abundant within these subsets in Ifnar1-/- than in wild-type mice. Moreover, blockade of IL-10R in Ifnar1-/- mice increased their susceptibility to S. typhimurium colitis, suggesting that altered numbers of these immunoregulatory cells may underlie the difference in disease severity. This cross-talk between type I IFN and IL-10R signaling pathways may represent a key host cellular mechanism to investigate further in order to unravel the balance between pathogenic inflammation and homeostasis of the colon. Taken together, our data clearly demonstrate that type I IFN signaling is pathogenic in S. typhimurium colitis.

The mammalian innate immune response is responsible for the early stages of defense against invading pathogens. One of the major receptor families facilitating innate immune activation is the Toll-like receptor (TLR) family. These receptors are type 1 membrane proteins spanning the membrane with a single transmembrane domain (TMD). All TLRs form homo- and hetero-dimers within membranes and new data suggest that the single transmembrane domain of some of these receptors is involved in their dimerization and function. Newly identified TLR dimers are continuously reported but only little is known about the importance of the TMDs for their dimer assembly and signaling regulation. Uncontrolled or untimely activation of TLRs is related to a large number of pathologies ranging from cystic fibrosis to sepsis and cancer. In this review we will focus on the contribution of the TMDs of innate immune receptors – specifically TLR2–to their regulation and function. In addition, we will address the current issues remaining to be solved regarding the mechanistic insights of this regulation. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)–derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

Protein–protein interactions within the membrane are involved in many vital cellular processes. Consequently, deficient oligomerization is associated with known diseases. The interactions can be partially or fully mediated by transmembrane domains (TMD). However, in contrast to soluble regions, our knowledge of the factors that control oligomerization and recognition between the membrane–embedded domains is very limited. Due to the unique chemical and physical properties of the membrane environment, rules that apply to interactions between soluble segments are not necessarily valid within the membrane. This review summarizes our knowledge on the sequences mediating TMD–TMD interactions which include conserved motifs such as the GxxxG, QxxS, glycine and leucine zippers, and others. The review discusses the specific role of polar, charged and aromatic amino acids in the interface of the interacting TMD helices. Strategies to determine the strength, dynamics and specificities of these interactions by experimental (ToxR, TOXCAT, GALLEX and FRET) or various computational approaches (molecular dynamic simulation and bioinformatics) are summarized. Importantly, the contribution of the membrane environment to the TMD–TMD interaction is also presented. Studies utilizing exogenously added TMD peptides have been shown to influence in vivo the dimerization of intact membrane proteins involved in various diseases. The chirality independent TMD–TMD interactions allows for the design of novel short d- and l-amino acids containing TMD peptides with advanced properties. Overall these studies shed light on the role of specific amino acids in mediating the assembly of the TMDs within the membrane environment and their contribution to protein function. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Cancer stem cells present a major therapeutic challenge - their effective eradication depends on therapeutic targeting of the relative vulnerabilities of the cancer stem cells vs. normal tissue stem cells. One feature that distinguishes cancer initiating and propagating cells from their normal counterparts is transcriptional addiction, providing opportunities for novel therapeutic interventions with the aim of curing cancer. CKIα ablation appears as a promising means of activating p53 and killing leukemia cells in MDS and AML (Elyada et al , doi:10.1038/nature09673; Kronke et al, doi: 10.1038/nature14610); yet, with no selective CKIα inhibitors for in vivo use, the therapeutic value of CKIα inhibition cannot be validated. We succeeded in developing two different classes of CKIα inhibitors, one inducing CKIα degradation, and another targeting the catalytic pocket of CKIα, with no degradation. Both inhibitor types activate p53, yet we found that CKIα ablation or degradation was not sufficient to selectively eliminate leukemia stem cells, due to robust expression of anti-apoptotic oncogenes driven by super-enhancer activation. Through extensive medicinal chemistry, we obtained CKIα catalytic inhibitors with profound killing capacity of primary mouse AML cells and human AML cell lines. We demonstrated a strong therapeutic effect in a mouse model of MLL-AF9-induced leukemia, with >40% cure rates evident by transplantation of bone marrow from treated leukemic mice to lethally irradiated normal mice. All transplanted mice regained normal blood counts, exclusively derived from the donor bone marrow. None showed any evidence of residual disease for 6 months, demonstrating a successful therapeutic window, distinguishing leukemia stem cells (LSCs) from HSPCs. Parallel in vitro experiments, show that the inhibitors did not affect normal hematopoietic progenitors in a CFU assay, but eliminated AML progenitors at an IC50 We found that CKIα inhibitors possessing potent anti-leukemia activity are distinguished by their capacity to co-target CDK9 and suppress the RNA Pol II elongation factor P-TEFb (CDK9-CyclinT1 complex), evident by blocking the phosphorylation of RNA Pol II C-terminal domain (pCTD2/5, see Figure). Co-crystallography studies elucidated the structural basis for co-targeting of CKIα and P-TEFb, the latter controls the rate limiting step of Poll-II-transcription and functions as a gatekeeper of super-enhancers. We found that both P-TEFb and super-enhancer activity are vigorously activated in AML cells. Therefore, the efficient P-TEFb-targeting activity of the inhibitors enables a selective blockade of leukemia super-enhancers (SE)-driven transcription, demonstrated by ChIP-seq analysis. As a result, SE-dependent transcription of major leukemia drivers including Myc and the anti-apoptotic oncogenes Bcl-2 and Mcl-1 was nearly abolished. Another remarkable feature of these dual CKIα-P-TEFb inhibitors is a rapid hit mechanism, by which, short drug exposure (10 mins in vitro and 2hrs in vivo) results in prolonged (24hrs) SE suppression and leukemia cell apoptosis. This unique property, which is at variance with the current occupancy-driven pharmacological paradigm, likely contributes to the dramatic therapeutic effect of co-targeting CKIα and P-TEFb in leukemia, whereby combination of rapid p53 induction with disruption of oncogenic super-enhancers eliminates leukemia stem cells without compromising normal HSPCs. The ability to disrupt super-enhancers and eliminate their associated oncogenes within minutes of drug exposure permits short intermittent therapy, which minimizes generalized toxicity. Indeed, at the therapeutic dose, we did not observe any significant adverse treatment effects even at daily treatment for 45 days in mice. To conclude, we developed a new class of small molecule inhibitors that co-targets CKIα and P-TEFb. These inhibitors have unique pharmacologic properties: short-term kinase inhibition results in long-term disruption of SE activity. Shutdown of leukemic super-enhancers in synergy with robust p53 activation compromises leukemic cells and stem cells addicted to SE-driven transcription. These features explain the powerful and specific anti-leukemic therapeutic effects of this new class of inhibitors in-vivo. Figure Figure. Disclosures Ben-Neriah: BioTheryX Inc.: Consultancy, Equity Ownership, Research Funding. Vacca: BioTheryX Inc.: Consultancy. Mercurio: BioTheryX: Employment, Equity Ownership.

The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine., IMPORTANCE The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification—Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal—has recently been reported in F. tularensis. We found that the ∆kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

Previously, we reported that intratumor or systemic inoculation of a cationic 15-mer, innate immunity-like lytic peptide composed of d- and l-amino acids ([D]-K6L9) caused growth arrest of 22RV1 prostate carcinoma xenografts in a mouse model. However, despite its therapeutic potential, this peptide has significant systemic toxicity at concentrations slightly higher than the therapeutic one. Here, we used the acidic environment created by solid tumors as a trigger to activate anticancer lytic peptides by making them cationic only at low pH levels. We achieved this selectivity by substituting lysines (pKa, ∼10.5) for histidines (pKa, ∼6.1) in the parental peptide [D]-K6L9. Histidine is protonated below pH 7. For that purpose, we replaced either three or all six lysines in the parental peptide with histidines to obtain the peptides [D]-K3H3L9 and [D]-H6L9. Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H6L9 mainly toward cancer cell lines. However, both peptides showed reduced systemic toxicity compared with the parental peptide. Intratumor and systemic inoculation of these peptides resulted in a significant decrease in the 22RV1 prostate cancer tumor volume and systemic secretion of prostate-specific antigen in a xenograft mice model. Moreover, histologic modifications revealed a significant reduction in new blood vessels selectively in tumor tissues after treatment with the peptides compared with the untreated tumors. The lytic mode of action of these new peptides, which makes it difficult for the cancer cells to develop resistance, and their selective and pH-dependent activity make them potential candidates for treatment of solid cancer tumors. [Cancer Res 2009;69(8):3458–63]

Toll Like Receptors (TLRs) are major facilitators of the innate immune response. Although the structure and function of TLRs are well studied, the role of their transmembrane domain has not been fully understood. Here we show that peptides based on the TM domain of TLR2 and TLR1/6 specifically inhibit TLR2 activation. These peptides inhibited TNFα secretion by mouse macrophages after lipotechoic acid (LTA, the cell wall of Gram-positive bacteria) activation, while no effect was seen on TLR4 activation by lipopolysacharide (LPS, the cell wall of Gram-negative bacteria). FRET analysis and self assembly assays revealed that these TM peptides are capable of interacting with each other, favoring hetero- rather than homo-dimerisation. Finally, a selected TLR2 TM peptide was able to rescue up to 60% of mice affected by an otherwise lethal acute systemic inflammation driven by hyperactivation of TLR2.