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  1. Author Response:

    Reviewer #1:

    The primary objective of this manuscript was to examine if multi-kinase inhibitor YKL-05-099 can inhibit salt inducible kinases (SIKs) with the goal to examine a new class of bone anabolic agents for the treatment of osteoporosis. They found that YKL-05-099 was successful in increasing anabolism and, surprisingly, decreasing bone resorption, leading them to investigate why this inhibitor differed from the effects of deletion of SIK2 and SIK3. They found that YKL-05-099 also inhibited the CSF1 (M-CSF) receptor, thus, inhibiting osteoclast activity. This is an interesting manuscript but there are some flaws in the conduct of the experiments and in the analyses which lessen its impact. Nevertheless, it opens the way for another possible oral therapeutic for osteoporosis.

    Reviewer #2:

    This work tests the ability of a kinase inhibitor to increase bone mass in a mouse model of osteoporosis. The inhibitor, which targets SIK and other kinases, was shown previously by these investigators to increase trabecular bone mass in young intact mice. Here they show that it increases trabecular, but not cortical, bone in oophorectomized mice and that this is associated with increased bone formation and little or no effect on bone resorption. In contrast, postnatal deletion of SIK2 and SIK3 increased both bone formation and resorption, suggesting that the inhibitor targets other kinases to control resorption. Indeed, the authors confirm that the inhibitor effectively suppressed the activity of CSF1R, a receptor tyrosine kinase essential for osteoclast formation. The authors also provide some evidence of unwanted effects of the inhibitor on glucose homeostasis and kidney function.

    Overall, the studies are performed well with all the necessary controls. The effects of the inhibitor on CSF1R inhibition are convincing and provide a compelling explanation for the net effects of the compound on the skeleton.

    1. The ability of the inhibitor to increase trabecular but not cortical bone mass will likely limit its appeal as an anabolic therapy. Indeed, the authors show that PTH, but not the inhibitor, increases bone strength. However, this limitation is not addressed in the manuscript. In addition, the mechanisms leading to these site-specific effects were not explored.

    We thank the reviewer for bringing up this important point. We have expanded the fifth paragraph of our discussion to include this important limitation, and to review potential mechanisms explaining this apparent compartment-selective of YKL-05-099:

    "Finally, our OVX study demonstrated that YKL-05-099 treatment increased trabecular, but not cortical, bone mass (Figure 1). In contrast, sclerostin antibody treatment increases both trabecular and cortical bone mass (58). At this point, we do not understand the mechanistic basis of this compartment-selective effect of this small molecule. Analysis of cortical bone at multiple time points after YKL-05-099 treatment is needed to conclusively demonstrate the absence of a cortical bone effect of this compound. However, Sik2/3 gene deletion appears to preferentially increase remodeling and bone formation on cancellous bone surfaces (Figure 4D). Therefore, it is possible that SIK inhibitors may stimulate remodeling-based bone formation. Further studies using larger animals with dedicated assays to measure modeling versus remodeling based bone formation (59) are needed to assess this possibility. Moreover, our current studies do not assess whether or not SIK inhibitors, like PTH or sclerostin antibody treatment, stimulate bone formation by activation of previously-quiescent bone lining cells (60, 61). The relative contribution of sclerostin suppression to YKL-05-099- mediated bone formation also remains to be determined."

    1. The mechanisms by which YKL-05-099 increases bone formation remain unclear. The authors point out that their previous studies indicate that the compound stimulates bone formation by suppressing expression of sclerostin. However, YKL-05-099 increased trabecular bone in the femur but not spine of intact mice and did not increase cortical bone in intact or OVX mice. In contrast, neutralization of sclerostin increases trabecular bone at both sites in intact mice as well as increases cortical bone thickness. These differences do not support the idea that YKL-05-099 increases bone formation by suppressing sclerostin.

    This is also a very important point, and has been addressed in the revised Discussion as detailed immediately above in point #1.

    1. The authors repeatedly state that the kinase inhibitor uncouples bone formation and bone resorption. However, the authors do not provide any direct evidence that this is the case. Although the term coupling is used to refer to a variety of phenomena in skeletal biology, the most common definition, and the one used in the review cited by the authors, is the recruitment of osteoblasts to sites of previous resorption. The authors certainly provide evidence that the kinase inhibitor independently targets bone formation and bone resorption, but they do not provide evidence that the mechanisms leading to recruitment of osteoblasts to sites of previous resorption has been altered. The resorption that takes place in the inhibitor-treated mice likely still leads to recruitment of osteoblasts to sites of resorption. Thus coupling remains intact.

    We thank the reviewer for raising this very important perspective. Here, we have relied upon serum bone turnover markers and histomorphometry on trabecular bone surfaces to measure bone formation and bone resorption. Others in the field have performed dynamic histomorphometry to assess calcein labeling adjacent to associated cement lines in order to distinguish between modeling and remodeling-based bone formation. Such analysis is operator dependent and nearly impossible to perform on trabecular surfaces in mice. The vast majority of published studies where this endpoint is reported are in non-human primates (ref 59) or human biopsy samples. Our claim here that YKL-05-099 treatment stimulates bone formation without increasing bone resorption is based upon the analytic methods that were used which demonstrate that this compound increases bone formation (BFR/BS by histomorphometry, osteoblast numbers by histomorphometry, and serum P1NP) without simultaneously stimulating bone resorption (osteoclast numbers and eroded surface by histomorphometry and serum CTX). Therefore, we have added the following new text in the Discussion:

    "Our current claim that YKL-05-099 ‘uncouples’ bone formation and bone resorption is based upon our histomorphometry and serum bone turnover marker data which clearly show that this agent increases bone formation without increasing the measured resorption-related parameters. Future study is needed using dynamic histomorphometry in association with cement line visualization (62) to see if this compound can stimulate modeling-based bone formation independent of bone resorption."

    1. The results of the current study nicely confirm previous findings by the same authors, demonstrating the reproducibility of the effects of the inhibitor. They also provide a compelling explanation for the net effect of the inhibitor on bone resorption (it stimulates RANKL expression but inhibits CSF1 action). While this latter finding will likely be of interest to those exploring SIK inhibitors for therapeutic uses, overall this study may be of limited appeal to a broader audience.

    We thank the reviewer for raising this point, and would like to take this opportunity to highlight the novel aspects of the current work:

    1. The first demonstration of dual kinase targeting by YKL-05-099 and the potential of this dual targeting to be exploited for therapeutic purposes in bone

    2. The first investigation of the effects of SIK inhibitor treatment in an osteoporosis disease model

    3. The first characterization of postnatal Sik2/3 mutant mice and demonstration that these animals only display obvious changes in bone biology

    4. Use of conformation-specific kinase modeling to demonstrate shared common features between how YKL-05-099 might engage two distinct kinases

    Given the dearth of novel bone anabolic targets, the enormous public health problem of osteoporosis, and the widespread interest in SIKs for multiple disease indications, we believe that the novel findings presented here are important for a broad audience.

    Reviewer #3:

    In this study, Tang and colleague report that the multikinase inhibitor YKL-05-099 increases bone formation and decreases bone resorption in hypogonadal female mice with mechanisms that are likely to involve inhibition of SIKs and CSFR1, respectively. The authors also report that postnatal mice with inducible, global deletion of SIK2 and SIK3 show an increase of bone mass that is associated to both an augmentation of bone formation and bone resorption.

    The paper provides novel and interesting information with potentially highly relevant translational implications. The quality of the data is outstanding and most of the authors' conclusions are supported by the data as shown.

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  2. Reviewer #3 (Public Review):

    In this study, Tang and colleague report that the multikinase inhibitor YKL-05-099 increases bone formation and decreases bone resorption in hypogonadal female mice with mechanisms that are likely to involve inhibition of SIKs and CSFR1, respectively. The authors also report that postnatal mice with inducible, global deletion of SIK2 and SIK3 show an increase of bone mass that is associated to both an augmentation of bone formation and bone resorption.

    The paper provides novel and interesting information with potentially highly relevant translational implications. The quality of the data is outstanding and most of the authors' conclusions are supported by the data as shown.

    Was this evaluation helpful?
  3. Reviewer #2 (Public Review):

    This work tests the ability of a kinase inhibitor to increase bone mass in a mouse model of osteoporosis. The inhibitor, which targets SIK and other kinases, was shown previously by these investigators to increase trabecular bone mass in young intact mice. Here they show that it increases trabecular, but not cortical, bone in oophorectomized mice and that this is associated with increased bone formation and little or no effect on bone resorption. In contrast, postnatal deletion of SIK2 and SIK3 increased both bone formation and resorption, suggesting that the inhibitor targets other kinases to control resorption. Indeed, the authors confirm that the inhibitor effectively suppressed the activity of CSF1R, a receptor tyrosine kinase essential for osteoclast formation. The authors also provide some evidence of unwanted effects of the inhibitor on glucose homeostasis and kidney function.

    Overall, the studies are performed well with all the necessary controls. The effects of the inhibitor on CSF1R inhibition are convincing and provide a compelling explanation for the net effects of the compound on the skeleton.

    1. The ability of the inhibitor to increase trabecular but not cortical bone mass will likely limit its appeal as an anabolic therapy. Indeed, the authors show that PTH, but not the inhibitor, increases bone strength. However, this limitation is not addressed in the manuscript. In addition, the mechanisms leading to these site-specific effects were not explored.

    2. The mechanisms by which YKL-05-099 increases bone formation remain unclear. The authors point out that their previous studies indicate that the compound stimulates bone formation by suppressing expression of sclerostin. However, YKL-05-099 increased trabecular bone in the femur but not spine of intact mice and did not increase cortical bone in intact or OVX mice. In contrast, neutralization of sclerostin increases trabecular bone at both sites in intact mice as well as increases cortical bone thickness. These differences do not support the idea that YKL-05-099 increases bone formation by suppressing sclerostin.

    3. The authors repeatedly state that the kinase inhibitor uncouples bone formation and bone resorption. However, the authors do not provide any direct evidence that this is the case. Although the term coupling is used to refer to a variety of phenomena in skeletal biology, the most common definition, and the one used in the review cited by the authors, is the recruitment of osteoblasts to sites of previous resorption. The authors certainly provide evidence that the kinase inhibitor independently targets bone formation and bone resorption, but they do not provide evidence that the mechanisms leading to recruitment of osteoblasts to sites of previous resorption has been altered. The resorption that takes place in the inhibitor-treated mice likely still leads to recruitment of osteoblasts to sites of resorption. Thus coupling remains intact.

    4. The results of the current study nicely confirm previous findings by the same authors, demonstrating the reproducibility of the effects of the inhibitor. They also provide a compelling explanation for the net effect of the inhibitor on bone resorption (it stimulates RANKL expression but inhibits CSF1 action). While this latter finding will likely be of interest to those exploring SIK inhibitors for therapeutic uses, overall this study may be of limited appeal to a broader audience.

    Was this evaluation helpful?
  4. Reviewer #1 (Public Review):

    The primary objective of this manuscript was to examine if multi-kinase inhibitor YKL-05-099 can inhibit salt inducible kinases (SIKs) with the goal to examine a new class of bone anabolic agents for the treatment of osteoporosis. They found that YKL-05-099 was successful in increasing anabolism and, surprisingly, decreasing bone resorption, leading them to investigate why this inhibitor differed from the effects of deletion of SIK2 and SIK3. They found that YKL-05-099 also inhibited the CSF1 (M-CSF) receptor, thus, inhibiting osteoclast activity. This is an interesting manuscript but there are some flaws in the conduct of the experiments and in the analyses which lessen its impact. Nevertheless, it opens the way for another possible oral therapeutic for osteoporosis.

    Was this evaluation helpful?
  5. Evaluation Summary:

    This is a very interesting, novel and informative study. The effects of the inhibitor on CSF1R inhibition are convincing and provide a compelling explanation for the net effects of the compound on the skeleton. The study opens the way for another possible oral therapeutic for osteoporosis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    Was this evaluation helpful?