Upregulating Wnt Signaling Through Twist1 Inhibition

[pegasus2]

.

 

[pegasus2]

Tissue-targeted R-spondin mimetics for liver regeneration​

R-spondins 1-4 (RSPO1-4) are a family of ligands that amplify Wnt signals through a receptor complex containing the zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43) proteins and the coreceptor leucine-rich repeat-containing G-protein coupled receptors 4-6 (LGR4-6)2,3,4. RSPOs contain two furin (Fu) repeats, Fu1 and Fu2, which in combination are sufficient to recapitulate Wnt signaling enhancing activity5. Fu1 primarily interacts with ZNRF3/RNF43 and Fu2 interacts with LGR4-65,6,7. ZNRF3 and RNF43 are membrane-bound E3 ligases that specifically target Wnt receptors (FZD1-10 and LRP5 or LRP6) for degradation8,9. Binding of RSPOs to ZNRF3/RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which stabilizes Wnt receptors and amplifies Wnt signaling. In addition, R-spondins might work through mechanisms that are independent of LGRs

RSPOs may be beneficial in adult tissue repair12,13, particularly in situations where the expression of endogenous Wnt ligands is upregulated but signaling (presumably limited by receptor stability) is insufficient to overcome tissue damage.

Catagen is initiated by a reduction of R-spondins mediated by FGFR, and Twist1 has been shown to regulate FGFR. If Twist1 is inhibited then r-spondins should be upregulated in the HF. R-spondins stabilize Wnt receptors which strongly upregulates Wnt signaling and maintains beta-catenin nuclear translocation, and induces hair growth.

Inhibiting Twist1 may upregulate R-spondins and RUNX2, and downregulate the AR. Unfortunately there are no selective and potent Twist1 antagonists on the market. This is where BAY and the PRLR might come into play. Twist inhibits apoptosis in cancer, yet it induces apoptosis in the HF. Prolactin works in the same way, it inhibits proliferation in the HF while promoting it in cancer. Inhibiting Twist1 or PRLR promotes hair growth and tumor regression. I'm speculating that hair growth from PRLR antagonists may be mediated, in part, through Twist1 inhibition. Prolactin induces STAT3/5 which upregulates Twist1. JAK inhibitors weren't tremendously effective for Androgenetic Alopecia when tested by Aclaris, but maybe targeting the JAK/STAT pathway wasn't entirely wrong. The hair cycle clock study shows that extended anagen "requires the presence of active Wnt signaling in the DP" in addition to fgfr2/3 ablation. It may be that in AA there is active Wnt signaling in the DP already, but not in Androgenetic Alopecia. PRLR antagonists may do something JAK inhibitors don't, such as upregulating RUNX2 and boosting Wnt activation in the DP. Tofacitinib downregulates RUNX2 instead.

Prolactin decreases expression of Runx2, osteoprotegerin, and RANKL in primary osteoblasts derived from tibiae of adult female rats

 

[pegasus2]

Novel Links among Wnt and TGF-β Signaling and Runx2

prostaglandin (PG)E2, which rapidly activates endogenous Runx2, modestly increased basal TCF/LEF-dependent gene expression, but it potently enhanced the stimulatory effect of gene induction by Wnt1 (Fig. 2A), or by LiCl and WAg (Fig. 2B). Notably, whereas activation of the TCF/LEF-sensitive promoter was significantly inhibited when endogenous Runx2 was suppressed by transgenic antisense expression (Fig. 2B, upper panel), this was particularly striking in PGE2-activated cells

Inasmuch as Runx2 and TCF/LEF exhibited codependent roles on gene expression in osteoblasts, we then asked whether the short-term Wnt induction protocol that we employed also modulated Runx2. In this regard, transgenic or soluble factor induction of the Wnt pathway for 24 h enhanced gene promoter activity by SXN1c or by the TβRI gene promoter (Fig. 2, E and F). However, it did not increase the total amount of nuclear Runx2 protein by Western blot analysis (upper panel, Fig. 2G), or by DNA binding (Fig. 2H) during this interval, even while it increased the amount of the Runx2-sensitive gene product TβRI by approximately 7-fold (middle panel, Fig. 2G), suggesting instead that it might increase the transcriptional potential of Runx2...Accordingly, transgenic expression of Wnt 1, which drives its effects through a Wnt receptor system that favors β-catenin stabilization (30), also did not directly enhance M1-Runx2 activity

Inasmuch as increases in expression or activation of endogenous Runx2 appeared to enhance gene expression through the Wnt pathway, and induction of the Wnt pathway was diminished by loss of endogenous Runx2, the increases or residual activities that we noted are likely dependent, at least in part, on the overall levels of Runx2.

Pharmacologic inhibition of JAK-STAT signaling promotes hair growth

Our findings demonstrate that inhibition of JAK-STAT signaling promotes hair growth by stimulating the activation and/or proliferation of HF stem cells, highlighting the role of this pathway in maintenance of HF quiescence. The observation that inhibition of JAK-STAT signals can promote activation or differentiation of stem/progenitor cells is not unique to the HF. Loss of Stat5 in hematopoietic stem cells induces exit from a quiescent state, leading to increased bone marrow–repopulating capacity after irradiation (47). Inhibition of JAK-STAT signaling improves skeletal muscle regeneration in aged mice by promoting symmetric satellite cell expansion and reduced commitment to myogenesis (48, 49). These findings are consistent with the association of JAK-STAT with quiescence in the HF (29) and with evidence for a role of Stat3 in progression of the normal hair cycle in adult mice (21, 22). Moreover, recent studies have shown that increased JAK-STAT signaling in aged mice inhibits HF stem cell function in vitro (50) and that Stat5 signaling controls HF stem cell quiescence during pregnancy and lactation (51). Therefore, the role of JAK-STAT signaling in promoting quiescence may represent a generalized mechanism in adult stem cell populations.

 

[pegasus2]

Potential Twist1 inhibitors

SH-4-54: A STAT3/5 inhibitor; so far it's effective in vitro against Glioma, breast cancer, and prostate cancer cell lines. Once again, all things that PRLR antagonists are effective against. It is shown to downregulate Twist.

Treatment of STAT3 inhibitor, SH-4-54, inhibited cell invasion, increased E-cadherin and downregulated N-cadherin, Twist and Survivin

https://pubmed.ncbi.nlm.nih.gov/32494165/

Harmine: It's been reported as the first Twist1 inhibitor, but it also inhibits LEF1 and TCF4. Those are targets of beta catenin so this isn't ideal. However, it comes from Syrian Rue which is used as a natural remedy for hair loss, so go figure. It's also a hallucinogen and used for abortion. It could be dangerous, especially when combined with other drugs.

Current efforts in our laboratory are ongoing to identify harmine analogues or related compounds that allow for more potent inhibition of TWIST1 transcriptional activity without associated neurotoxicity.

In summary, we identified harmine as a first-in-class inhibitor of TWIST1 with broad cytotoxic activity in the three major classes of oncogene-driven NSCLC, EGFR-mutant, KRAS mutant, and c-MET-amplified/mutant. Given that we have previously established the requirement of TWIST1 for tumorigenesis in oncogene-driven lung cancer with these genetic backgrounds (10), using harmine derivatives may be a viable therapeutic option to treat oncogene-driven NSCLC both in the treatment-naïve and acquired resistance setting. Additionally, as TWIST1 is rarely expressed post-natally (45,46), pharmacological inhibition of TWIST1 may be associated with minimal side-effects.

AGX51: https://www.biorxiv.org/content/10.1101/2020.01.06.894840v1.full This is an Id antagonist, so again not good for hair. Ids are required to prevent stem cell depletion, and their knockout prevents hair growth after several cycles in mice.

BMP Signaling and Its pSMAD1/5 Target Genes Differentially Regulate Hair Follicle Stem Cell Lineages

This inverse correlation between IDs and proliferation became more pronounced during repetitive rounds of depilation-induced hair cycling, until eventually Id1 cKO animals failed to regrow their hair coat (Figure 5H). Concomitantly, the numbers of cells expressing HFSC markers waned (Figure 5I). These results suggested that ID proteins function to maintain the HFSC pool by restricting their proliferation and wasteful usage.

Quercetin: Quercetin restrains TGF-β1-induced epithelial–mesenchymal transition by inhibiting Twist1

 

[pegasus2]

There is a recent patent out of UCLA for siRNA and small molecule inhibitors of Twist for cancer treatment. They also have another patent for peptide inhibitors of Twist. Our best hope may be that they update the patent with a specific small molecule antagonist of Twist1. They do have a list of PI3K inhibitors in the first patent that inhibit Twist1, but those wouldn't be beneficial in our case.

Targeting Twist expression with small molecules(Pei, Haixiang et al.) This paper lists some more compounds that target twists, but some like tamoxifen cause hair loss due to their other actions. It also says that upregulating miR-548c could downregulate Twist. One compound that is said to grow hair is thymoquinone. Another, Berberine hydrochloride, is known to induce hypertrichosis. None of these are as potent as harmine, but may be better if they don't have other harmful effects.


More on Twist1 interactions in the hair follicle

Differential Expression between Human Dermal Papilla Cells from Balding and Non-Balding Scalps Reveals New Candidate Genes for Androgenetic Alopecia - ScienceDirect

we found TWIST1, a DP signature gene expressed in both BAB and BAN (Figure 1e and see Supplementary Table S1a), being up-regulated in BAB[balding men] (Table 1). Twist1, a basic helix-loop-helix protein, is crucial for anagen-to-catagen transition during the hair growth cycle with Twist1 protein ablation in adult mice DP resulting in prolonged anagen (Xu et al., 2013). Hence, TWIST1 up-regulation in DPCs of balding scalps compared to non-balding scalps may result in accelerated transition from anagen to catagen and thus a shortened period of anagen during the hair cycle, a phenomenon in balding scalps that leads to the formation of short vellus hairs instead of long terminal hairs (Paus and Cotsarelis, 1999).

TWIST1 also interacts and binds to HDAC4 (Danciu and Whitman, 2009) to regulate gene expression (Gong and Li, 2002, Lee et al., 2003). In addition, binding of TWIST1 at E-boxes in the AR promoter region results in up-regulated AR expression

 

[pegasus2]

any comments on using JAK? my only criteria are toxicity and bone growth when selecting a WNT agonist...

Safety profile of baricitinib for the treatment of rheumatoid arthritis over a median of 3 years of treatment: an updated integrated safety analysis - The Lancet Rheumatology

Lilly’s baricitinib meets primary endpoint in alopecia areata trial (clinicaltrialsarena.com)

JAK inhibition increases bone mass in steady-state conditions and ameliorates pathological bone loss by stimulating osteoblast function - PubMed (nih.gov)

JAK inhibitors haven't shown to be as effective for male pattern baldness as for other types of hair loss. They downregulate RUNX2 and ID proteins. A selective and potent STAT inhibitor like SH-4-54 combined with a potent Wnt agonist like KY19382 might be better than a JAK inhibitor. If you were going to try a JAK inhibitor I'd combine it with KY19382.

 

[BaldAndBalder]

Can someone make a tl;dr for a brainlet like myself?