Multiple sclerosis (
MS
) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (
HA
) and its receptors have been implicated in disease pathogenesis.
HA
...exerts its effects through four different
G
protein‐coupled receptors designated
H
1
‐
H
4
. We previously examined the effects of traditional single
HA
receptor (
HR
) knockouts (
KO
s) in experimental allergic encephalomyelitis (
EAE
), the autoimmune model of
MS
. Our results revealed that
H
1
R
and
H
2
R
are propathogenic, while
H
3
R
and
H
4
R
are antipathogenic. This suggests that combinatorial targeting of
HR
s may be an effective disease‐modifying therapy (
DMT
) in
MS
. To test this hypothesis, we generated
H
1
H
2
RKO
and
H
3
H
4
RKO
mice and studied them for susceptibility to
EAE
. Compared with wild‐type (
WT
) mice,
H
1
H
2
RKO
mice developed a less severe clinical disease course, whereas the disease course of
H
3
H
4
RKO
mice was more severe.
H
1
H
2
RKO
mice also developed less neuropathology and disrupted blood brain barrier permeability compared with
WT
and
H
3
H
4
RKO
mice. Additionally, splenocytes from immunized
H
1
H
2
RKO
mice produced less interferon(IFN)‐γ and interleukin(IL)‐17. These findings support the concept that combined pharmacological targeting of
HR
s may be an appropriate ancillary
DMT
in
MS
and other immunopathologic diseases.
Acquired secondary resistance mutations in FLT3 have been shown to limit the therapeutic benefit of FLT3 inhibitors in FLT3-ITD mutated AML. Multiple strategies have been pursued to address such ...resistance, including the development of kinase inhibitors that use alternate binding modes or by simultaneously targeting additional pathways. This latter approach may be focused on suppression of parallel oncogenic pathways to treat the acquired resistance, or on strategies to reduce or delay the acquisition of resistance. Here, we describe a rationally conceived next generation FLT3 inhibitor, FLX925, that was prospectively designed to address or avoid common resistance mechanisms with a unique binding mode and potent activity against CDK4/CDK6. We contextualize our findings by comparing FLX925 to other FLT3 inhibitors (quizartinib and gilteritinib) currently in late-stage clinical development and demonstrate that FLX925 has a superior resistance profile.
FLX925 is a potent and selective type-1 inhibitor of FLT3 that retains its cellular potency against clinically relevant secondary resistance mutations in FLT3. This was evaluated in multiple experimental systems including isogenic Ba/F3 cells engineered to express FLT3-ITD with, or without, various known secondary FLT3 mutations. In addition, these data were extended to the human setting using both the MOLM13 and MOLM14 FLT3-ITD mutated AML cell lines and subclones of these lines harboring well-characterized resistance mutations described elsewhere. Data have been published previously demonstrating a superior resistance profile for FLX925 when compared to quizartinib and sorafenib. When compared to gilteritinib, FLX925 had a favorable profile of relative potencies against a range of FLT3-ITD resistance mutations.
In addition to the isogenic murine and human models of FLT3 inhibitor resistance, with engineered known genetic alterations, we explored the activity of FLX925 and other FLT3 inhibitors in a model of in vitro acquired resistance. Using MOLM13 cells and standard protocols for the generation of drug resistance, we demonstrate that the magnitude of resistance to quizartinib and gilteritinib greatly exceeds that observed with FLX925 during the same timeframe. Pools of resistant cells from each compound treatment are being analyzed by next-generation sequencing in an effort to better understand the mechanism of resistance associated with each compound. We hypothesize that the CDK4/6 inhibitory activity, which is unique to FLX925, contributes to its superior resistance profile. Whether this is solely linked to an impact on the cell cycle or more recent findings of CDK6-driven transcription of FLT3 and PIM1 is an active area of investigation. Nonetheless, the CDK4/6 activity of FLX925 potentially broadens the utility of this compound to FLT3 wild-type AML. Here, we show FLX925, in contrast to other FLT3 inhibitors, potently suppresses the proliferation of a panel of AML cell lines. These data have been extended to FLT3 wild-type AML patient samples in which FLX925, but not gilteritnib, induced a desirable pharmacodynamic effects. The totality of the preclinical data suggest FLX925 may be a best-in-class inhibitor for the treatment of AML with, or without, FLT3-ITD mutations. FLX925 is currently being investigated in a Ph1/b dose-escalation study in subject with relapsed or refractory AML (NCT02335814).
Marubayashi:FLX Bio: Employment, Equity Ownership. Park:FLX Bio: Employment, Equity Ownership. Noubade:FLX Bio: Employment, Equity Ownership. Phan:FLX Bio: Employment. Cutler:FLX Bio: Employment, Equity Ownership; Amgen, Inc: Equity Ownership. Kassner:FLX Bio: Employment, Equity Ownership. Fridman:FLX Bio: Employment, Equity Ownership.
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts ...its effects through four different G protein‐coupled receptors designated H1‐H4. We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H1R and H2R are propathogenic, while H3R and H4R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease‐modifying therapy (DMT) in MS. To test this hypothesis, we generated H1H2RKO and H3H4RKO mice and studied them for susceptibility to EAE. Compared with wild‐type (WT) mice, H1H2RKO mice developed a less severe clinical disease course, whereas the disease course of H3H4RKO mice was more severe. H1H2RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H3H4RKO mice. Additionally, splenocytes from immunized H1H2RKO mice produced less interferon(IFN)‐γ and interleukin(IL)‐17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.
Multiple sclerosis is a sexually dimorphic, demyelinating disease of the CNS, and experimental allergic encephalomyelitis (EAE) is its principal autoimmune model. Young male SJL/J mice are relatively ...resistant to EAE whereas older males and SJL/J females of any age are susceptible. By comparing a wide age range of proteolipid protein peptide 139-151 immunized mice, we found that female disease severity remains constant with age. In contrast, EAE disease severity increases with age in SJL/J males, with young males having significantly less severe disease and older males having significantly more disease than equivalently aged females. To determine whether the Y chromosome contributes to this sexual dimorphism, EAE was induced in consomic SJL/J mice carrying a B10.S Y chromosome (SJL.Y(B10.S)). EAE was significantly more severe in young male SJL.Y(B10.S) mice compared with young male SJL/J mice. These studies show that a Y chromosome-linked polymorphism controls the age-dependent EAE sexual dimorphism observed in SJL/J mice.
Histamine H(3) receptor (Hrh3/H(3)R) is primarily expressed by neurons in the central nervous system (CNS) where it functions as a presynaptic inhibitory autoreceptor and heteroreceptor. Previously, ...we identified an H(3)R-mediated central component in susceptibility to experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS), related to neurogenic control of blood brain barrier permeability and peripheral T cell effector responses. Furthermore, we identified Hrh3 as a positional candidate for the EAE susceptibility locus Eae8. Here, we characterize Hrh3 polymorphisms between EAE-susceptible and resistant SJL and B10.S mice, respectively, and show that Hrh3 isoform expression in the CNS is differentially regulated by acute peripheral inflammatory stimuli in an allele-specific fashion. Next, we show that Hrh3 is not expressed in any subpopulations of the immune compartment, and that secondary lymphoid tissue is anatomically poised to be regulated by central H(3)R signaling. Accordingly, using transcriptome analysis, we show that, inflammatory stimuli elicit unique transcriptional profiles in the lymph nodes of H(3)RKO mice compared to WT mice, which is indicative of negative regulation of peripheral immune responses by central H(3)R signaling. These results further support a functional link between the neurogenic control of T cell responses and susceptibility to CNS autoimmune disease coincident with acute and/or chronic peripheral inflammation. Pharmacological targeting of H(3)R may therefore be useful in preventing the development and formation of new lesions in MS, thereby limiting disease progression.
Histamine (HA), a biogenic amine with a broad spectrum of activities in both physiological and pathological settings, plays a key regulatory role in experimental allergic encephalomyelitis, the ...autoimmune model of multiple sclerosis. HA exerts its effect through four G protein-coupled receptors designated HA receptor H 1 , H 2 , H 3 , and H 4 . We report here that, compared with wild-type animals, mice with a disrupted HA H 3 receptor (H3RKO), the expression of which is normally confined to cells of the nervous system, develop more severe disease and neuroinflammation. We show that this effect is associated with dysregulation of blood–brain barrier permeability and increased expression of MIP-2, IP-10, and CXCR3 by peripheral T cells. Our data suggest that pharmacological targeting of the H 3 R may be useful in preventing the development and formation of new lesions in multiple sclerosis, thereby significantly limiting the progression of the disease.
Disruption of the blood–brain barrier (BBB) underlies the development of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Environmental factors, such as
Bordetella pertussis
, ...are thought to sensitize central endothelium to biogenic amines like histamine, thereby leading to increased BBB permeability.
B. pertussis
-induced histamine sensitization (Bphs) is a monogenic intermediate phenotype of EAE controlled by histamine H
1
receptor (
Hrh1
/H
1
R). Here, we transgenically overexpressed H
1
R in endothelial cells of
Hrh1
-KO (H
1
RKO) mice to test the role of endothelial H
1
R directly in Bphs and EAE. Unexpectedly, transgenic H
1
RKO mice expressing endothelial H
1
R under control of the von Willebrand factor promoter (H
1
RKO-vWF
H1R
Tg) were Bphs-resistant. Moreover, H
1
RKO-vWF
H1R
Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H
1
RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H
1
R expression reduces BBB permeability, suggesting that endothelial H
1
R signaling may be important in the maintenance of cerebrovascular integrity.
Abstract
T helper (TH) cells modulate immune responses to better cope with various types of invading pathogens through the production of different effector cytokines. TH17 cells produce ...Interleukin(IL)-17, and IL-22, and play an indispensable role in host defense against infections of extracellular pathogens. IL-22, a member of the IL-10 cytokine family, enhances proinflammatory innate defense mechanisms from epithelial cells, and provides crucial tissue protection from damages caused by inflammation and infection. Transforming growth factor (TGF)-β differentially regulates IL-22 and IL-17 expression. IL-6 alone promotes T cells to only produce IL-22, whereas the combination of IL-6 with high concentrations of TGF-β results in production of IL-17 but not IL-22. Here we identify the transcription factor c-Maf, which is induced by TGF-β, as a downstream repressor of Il22. c-Maf binds to the Il22 promoter, and is both necessary and sufficient for the TGF-β-dependent suppression of IL-22 production in TH17 cells. While a number of factors, such as RORγt, AhR and BATF, have been identified as positive regulators of IL-22 expression, c-Maf is the first transcription factor shown to be involved in its negative regulation. This is even more surprising since c-Maf at the same time is critical in promoting the expression IL-10 and IL-21 in T cells.
Abstract
The histamine H1 receptor (Hrh1/H1R) was identified as a shared autoimmune disease (SAID) gene in experimental allergic encephalomyelitis (EAE) and autoimmune orchitis, the principal AI ...models of multiple sclerosis (MS) and idiopathic male infertility, respectively. As a SAID gene, Hrh1/H1R can exert effects in multiple cell types including endothelial cells, T cells, and antigen presenting cells at critical check points during both the induction and effector phases of disease. In this regard, we showed that selective re-expression of H1R by endothelial cells in Hrh1-KO (H1RKO) mice significantly reduced disease severity whereas H1R expression by H1RKO T cells complemented EAE severity and cytokine responses. Given that the H1R has been reported to influence innate immune cell maturation, differentiation, chemotaxis, and cytokine production, which in turn influences CD4+ T cell effector responses, we selectively re-expressed H1R in CD11b+ myeloid cells of H1RKO mice to test the hypothesis that H1R signaling in these cells contributes to EAE susceptibility and/or T cell effector responses. We demonstrate that transgenic re-expression of H1R by H1RKO-CD11b+ cells neither complements EAE susceptibility nor T cell cytokine responses. These results further highlight the cell-specific effects that an AID gene can play in the pathogenesis of complex diseases such as EAE and MS, and the need for cell-specific targeting in optimizing therapeutic interventions based on such genes.
Histamine receptor H1 (H1R) is a susceptibility gene in both experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune orchitis (EAO), 2 classical T cell-mediated models of ...organ-specific autoimmune disease. Here we showed that expression of H1R in naive CD4+ T cells was required for maximal IFN-gamma production but was dispensable for proliferation. Moreover, H1R signaling at the time of TCR ligation was required for activation of p38 MAPK, a known regulator of IFN-gamma expression. Importantly, selective reexpression of H1R in CD4+ T cells fully complemented both the IFN-gamma production and the EAE susceptibility of H1R-deficient mice. These data suggest that the presence of H1R in CD4+ T cells and its interaction with histamine regulates early TCR signals that lead to Th1 differentiation and autoimmune disease.
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