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.

Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis.

Francisella tularensis is the causative agent of tularemia, a category A bioterrorism agent. The lipopolysaccharide (LPS) O antigen (OAg) of F. tularensis has been considered for use in a glycoconjugate vaccine, but conjugate vaccines tested so far have failed to confer protection necessary against aerosolized pulmonary bacterial challenge. When F. tularensis OAg was purified under standard conditions, the antigen had a small molecular size [25 kDa, low molecular weight (LMW)]. Using milder extraction conditions, we found the native OAg had a larger molecular size [80 kDa, high molecular weight (HMW)], and in a mouse model of tularemia, a glycoconjugate vaccine made with the HMW polysaccharide coupled to tetanus toxoid (HMW-TT) conferred better protection against intranasal challenge than a conjugate made with the LMW polysaccharide (LMW-TT). To further investigate the role of OAg size in protection, we created an F. tularensis live vaccine strain (LVS) mutant with a significantly increased OAg size [220 kDa, very high molecular weight (VHMW)] by expressing in F. tularensis a heterologous chain-length regulator gene (wzz) from the related species Francisella novicida Immunization with VHMW-TT provided markedly increased protection over that obtained with TT glycoconjugates made using smaller OAgs. We found that protective antibodies recognize a length-dependent epitope better expressed on HMW and VHMW antigens, which bind with higher affinity to the organism.

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.

Type I interferon signaling restrains IL-10R+ colonic macrophages and dendritic cells and leads to more severe Salmonella colitis.

Type I interferons (IFNα, IFNß) 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 IFNß- 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.

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19.

UNLABELLED: Differences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background. IMPORTANCE: Francisella tularensis is a highly pathogenic bacterium with a very low infectious dose in humans. Some mechanisms of bacterial virulence have been elucidated, but the host genetic factors that contribute to host resistance or susceptibility are largely unknown. In this work, we have undertaken a genetic approach to assess what these factors are in mice. Analyzing early interactions of macrophages with the bacteria as well as data on overall susceptibility to infection revealed a locus on chromosome 19 that is associated with both phenotypes. In addition, our work revealed differences in the early macrophage response between macrophages with different genetic backgrounds. Overall, this work suggests some intriguing links between in vitro and in vivo infection models and should aid in further elucidating the genetic circuits behind the host response to Francisella tularensis infection.

Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.

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-1ß. 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.

Regulation of innate immune responses by transmembrane interactions: lessons from the TLR family.

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.

Assembly of the TLR2/6 transmembrane domains is essential for activation and is a target for prevention of sepsis.

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.

Kdo hydrolase is required for Francisella tularensis virulence and evasion of TLR2-mediated innate immunity.

UNLABELLED: 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 (LD(50)) 2 log(10) units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) 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-1ß 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.

Transmembrane domains interactions within the membrane milieu: principles, advances and challenges.

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.

Suppression of human solid tumor growth in mice by intratumor and systemic inoculation of histidine-rich and pH-dependent host defense-like lytic peptides.

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]-K(6)L(9)) 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, approximately 10.5) for histidines (pKa, approximately 6.1) in the parental peptide [D]-K(6)L(9). 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]-K(3)H(3)L(9) and [D]-H(6)L(9). Interestingly, in vitro experiments showed pH-dependent activity only with [D]-H(6)L(9) 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.