Peer review of: Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B- (2019)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Dear Nature Communications, 

The manuscript entitled “Btk SH2-kinase interface is critical for allosteric kinase activation and its 
targeting inhibits B-cell neoplasms” by Duarte and colleagues describes the finding of an allosteric site 
on the kinase Btk that is required for it to become activated and the development of a repebody to 
bind to that region. Btk is the target of the drug ibrutinib which is a widely used and effective 
treatment for B-cell cancers and is therefore of significant pharmacological interest. Although ibrutinib 
is highly effective, resistance to therapy can arise by mutation of the kinase and therefore different 
modes of targeting the molecule are of interest. The identification of an allosteric site in this 
manuscript could aid such an approach. 

The authors describe some MD simulations which suggest a loose association with the back-side of the 
SH2 domain and the N-lobe of the kinase. This side of the SH2 domain is the site of several disease-
causing mutations in BTK which suggests a functional consequence. These mutations lead to a loss of 
kinase activation whilst they do not impair phosphopeptide binding. SAXS analysis by the authors 
suggests that the kinase can exist in an extended conformation and surmise that this is necessary for 
activation. Finally they develop a protein that binds to the allosteric site and show in cells and in vitro 
that it can inhibit autoactivation. This suggests that this site may be a genuine target for drug 
development 

This is a very detailed manuscript with an extremely impressive amount of experimental data. 
Although there is no “killer” experiment that proves the existence of this allosteric site there is a 
sufficient quantity of supporting data to imply that it exists. Of course, the existence of an allosteric 
site would be a lot more convincing if there were mutations in the predicted site on the N-lobe of the 
kinase that were made that interfered with activation, however that could prove to be a very difficult 
undertaking and I don’t suggest that it be required for publication. My recommendation is that the 
manuscript is of sufficient interest and novelty to be published in Nature Communications. 

Comments: 

Results: line 132: The authors state that stability is unaffected however in Supp Fig 1 it is clear that 4 
of the 5 mutants have a decreased thermal stability of 5-6 degrees which is actually quite significant 
for a single point mutation. 

Results: line 222: I’m not sure what this sentence means. Do the authors mean that the linker was 
removed and MD performed on the SH2 and KD domains as separate chains? If so, it is not surprising 
that the interaction didn’t occur as tethering of two domains vastly increases their chance of 
interacting and does not point to individual residues in the linker interacting directly. 

Results: line 237: “excellent agreement” is an overstatement for the sh3-sh2-kd data 

Results: line 256: Both K296E and S371P are disease causing mutations in Btk however they have 
opposite effects on autophosphorylation-do the authors have an explanation for this? 

Results: line 337: The interpretation of the SAXS data indicating a disruption in the interdomain 
interface is very tenuous. The SAXS-based reconstructions are of too low a resolution to “indicate” 
such a thing, maybe “suggest” is a more appropriate verb. 

Results: Line 344: This is an important result as it shows that the rF10 site is not “hidden” in longer 
forms of the protein – have the authors performed the ITC experiment more than once? 

Results: line 366: Was this with btk that was pre-auto-activated? If so its quite a surprising result. 

Results: line 396: “..rF10 was able to decrease pY551 of different C481S constructs”. Is that true? 
Figure 7b says only the SH2-KD phosphorylation was significantly inhibited 

Minor comments: 

Intro: line 83: How common is resistance to ibrutinib? 

Reviewer #2 (Remarks to the Author): 

This manuscript describes the use of a battery of methods and a set of mutants to decipher the 
domain interaction in BTK and suggests that peptides may be used to interfere with the SH2 and 
kinase domains, including serving as inhibitors for this kinase. It also contains a novel structure for 
the BTK SH2 domain. 

The first part is clear although there are certain issues deserving comments: 

1. In the ABSTRACT line 30, it is stated “…constitutive BTK signaling drives several B-cell 
neoplasms…”. For tumors responding to BTK inhibitors (BTKi), only rarely is constitutive BTK signaling 
a driver; rather tumor cells, similar to normal B-cells, are addicted to BTK for the adhesive processes 
necessary for these cells to remain in lymphoid organs, where they thrive. 

2. In the INTRODUCTION, line 76 & continued, I do not find that the history of BTKi development is 
correctly described. As an example, the BTKi, which later was named ibrutinib, was developed early on 
and published already in 2007 by Pan Z et al., and this paper is not cited. 

3. Fig. 1f. BTK WT’s relative pY551 could be 100 or 1 rather than around 75. 

4. Line 325, the fold increase seems more like ~6.5 rather than ~10. 

The second part, aiming at demonstrating that repebody protein, rF10, works as a BTKi, is more 
problematic, although from Fig. 6, panel a, rF10-induced expression seems to negatively influence the 
survival of HBL-1 cells. 

The basis for BTK activation is that SRC-family kinases like LYN phosphorylates BTK at Y551 (the 
classical initial activation step for all cytoplasmic protein-tyrosine kinases) and subsequently the main 
BTK substrate PLCG2 is phosphorylated. Since BTK also can be its own substrate, pY223 is frequently 
used as a measure of BTK’s catalytic activity. When inhibitors are used it is the effect on BTK Y223 
and PLCG2, which is the predominant readout. The authors of this manuscript instead seem to 
concentrate on the effect on pY551 (e.g. Fig. 6 d). The pY223 level is severely lowered by ibrutinib for 
the HBL-1 cell line in Fig. 6, panel c (this control is missing from the DOHH2 cells in Fig. 6, panel e), 
whereas rF10 seems to have a more modest effect and the same is true also for the PLCG2 substrate, 
as measured by pY1217 and pY753. 

ERK phosphorylation, which is a downstream in the B-cell receptor signaling pathway and representing 
an indirect measure of BTK enzyme function, seems essentially unaffected in Fig. 6, panel c, whereas 
a very potent effect is noticed in Fig. 6, panel e (i.e. in spite of rather weak activity on BTK Y223 and 
pY753/1217 in PLCG2, vide supra). This result could be interpreted as if rF10 has an effect on the 

phosphorylation of ERK in DOHH2 cells, which is BTK-independent; moreover, such an effect was not 
obvious in the HBL-1 cells. Collectively, while the authors claim effects on BTK pY223 and PLCG2 
pY753/pY1217 these effects are not as visible as the one on BTK pY551. 

To complement these figures there is also supplementary material. BTK inhibition can impair survival 
and cell division of sensitive target B-cells. This is stated by the authors, but from e.g. Supplementary 
Fig. 6 b this is not obvious – is it a single measurement which is statistically different? 

A possible way to study this would be to combine e.g. ibrutinib treatment and rF10-induced expression 
in some form of titration experiment. 

Minor points: 
Line 161 resembles (not resemble) 
Line 466 provides (not provide) 
Line 490 DNase (not DNAse) 
Line 715 Dawn Heleos (not Dawan Hellios) 

Some sentences need improvement/correction 

Reviewer #3 (Remarks to the Author): 

In this manuscript, Duarte et al. provide biophysical and biochemical evidence that the SH2 domain of 
Btk plays a critical role in the activation of its kinase domain, and that a subset of loss-of-function Btk 
mutations in the SH2 domain that have been linked to human X-linked agammaglobulinemia (XLA) are 
due to loss of this role. In addition, the authors have developed a repebody (LRR protein) that binds 
with high affinity to the Btk SH2 domain and inhibits Btk kinase activity in vitro and in cells, evidently 
by abrogating the stimulatory SH2-kinase interaction. A crystal structure of the repebody with the SH2 
domain was determined to understand the precise binding mode. The repebody was tested in vitro 
and in several cell lines and found to inhibit Btk autophosphorylation and signaling. This study will be 
of strong interest to those in the protein kinase and kinase therapeutics fields. 

Major concern: 

• A major aspect of this story is that the Btk SH2 domain binds to the N lobe of the kinase domain to 
stimulate tyrosine kinase activity, yet the authors tested only one mutation in the SH2 domain, S371P 
(an XLA mutation), to probe the putative SH2-kinase interface suggested by the MD simulations and 
SAXS data. Other mutations in the putative interface should be tested, irrespective of whether they 
are XLA mutations. Better yet would be the design of a gain-of-function point mutation in the SH2 
domain (in the putative interface), which leads to increased phosphorylation of Y551 relative to wild-
type. This should be possible, given that the SH2-kinase stimulatory interaction appears to be quasi-
stable, i.e., there’s room for improvement. 

Minor point: 
• The Introduction (first part) could be shortened; a review of protein kinases as targets of small-
molecule inhibitors is not necessary.