Saracatinib and Dasatinib Fail To Prevent Heritable Pulmonary Arterial Hypertension

Evidence suggests that the deregulation of SRC Family Kinases may play a role in the development of heritable pulmonary arterial hypertension, associated with BMPR2 mutations. The truncated c-terminus of the BMPR2 protein is known to increase the phosphorylation and downstream activity of SRC tyrosine kinases. To test the hypothesis that the inhibition of SRC can prevent heritable PAH due to a BMPR2 mutation, we exposed BMPR2 mutant mice to SRC inhibitors, saracatinib and dasatinib, to block the SRC activation caused by the BMPR2 mutation. Saracatinib and dasatinib failed to prevent the development of PAH in BMPR2 mutant mice. Increased pressure in the right ventricle was not normalized and muscularization of large blood vessels was not reduced when compared to wild type mice. Inhibiting SRC’s phosphorylation does not prevent heritable PAH, and thus supports evidence that SRC’s aberrant localization and trafficking in PAH plays a more critical role in disease development.

Pulmonary arterial hypertension (PAH) is a progressive and fatal illness of the 64 pulmonary microvasculature. The remodeling of the small resistance arteries in the lung 65 leads to increased pulmonary vascular resistance, increasing the workload on the heart's 66 right ventricle (RV) until it eventually fails (1,2).

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The most studied heritable risk factor for the development of PAH is a mutation 69 in the type 2 receptor in the BMP pathway (BMPR2). However, the mechanism 70 underlying BMPR2 and the development of PAH is not well understood (3-6 surgical anesthesia (Avertin) and the RVSP was measured by inserting a catheter into the 116 right heart through the right jugular vein in a closed-chested procedure, as previously 117 described (17). Ten-second segments of RVSP measurement were extracted from the 118 RVSP waveform, and the maximum RVSP was calculated by measuring the difference 119 between the peak and trough of the wave.  associated with multiple molecular abnormalities including increased SRC phosphorylation.

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To determine whether inhibition of this SRC activity would prevent pulmonary 157 hypertension, age and sex matched wild type and ROSA26-BMPR2 R899X mutant mice were 158 treated with saracatinib and dasatinib for the last four weeks during six weeks of transgene 159 activation. Saracatinib and dasatinib were shown to inhibit the action of downstream targets in the 160 SRC pathways (Figure 1), indicating proper osmotic delivery through the subcutaneously placed 161 pumps to achieve direct SRC inhibition.

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While vehicle-treated mice developed elevated RVSP at about 35% penetrance, mice 163 treated with SRC inhibitors have pressures indistinguishable from the vehicle-treated BMPR2 164 mutants (Figure 2). BMPR2 mutant mice have greater RVSPs than WT mice (p<0.05) and 165 delivery of saracatinib and dasatinib to mutant mice does not reduce RVSP when compared 166 vehicle treated mutants (p>0.05).

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Lung sections from ROSA26-BMPR2 R899X mutant mice had an increased number of 168 large muscularized vessels when compared to the WT mice (p<0.05) (Figure 3). The number of 169 large muscularized vessels in the mutant mice was not reduced to the wild type mice by 170 saracatinib and dasatinib treatment (p>0.05). The number of fully muscularized vessels is about 171 doubled in all BMPR2 mutant mice, independent of treatment. All vessels were consistently 172 measured along the longest axis and included the border of the masculinized vessels. Fully 173 muscularized vessels were measured and vessels that did not have at least 75% vessel perimeter 174 were excluded from the analysis. The muscles were stratified within Nikon and analyzed within 175 R. All imaging channels were taken manually and overlaid in ImageJ.  However, a key difference is that this previous study also aimed to restrict the aberrant SRC 207 trafficking seen in mutant cells. Therefore, it is possible that the differential trafficking of SRC in 208 heritable PAH plays a significant role in disease development and the inhibition of SRC itself is 209 only part of the strategy that would need to be adopted the treat the disease. SRC is known to 210 be sequestered in its phosphorylated form in PAH, and is thus unable to activate distant targets. 211 This sequestration may be the dominating problem in SRC's role in PAH, and inhibiting SRC's 212 phosphorylation may be the incorrect target to prevent heritable PAH (14). Interestingly, one of 213 the therapies, dasatinib, is known to cause PAH in humans (19). This clinical observation further 214 bolsters our belief that inhibiting SRC does not prevent disease, and the localization and 215 trafficking is a more likely player in pathogenesis. Our study indicates that more information is   have about twice as many fully muscularized large sized vessels (50-100um). Saracatinib and dasatinib administration did not normalize the number of large muscularized vessels in the mutant mice. The error bars are reported as SEM. B) Representative images of the ROSA26-BMPR2 R899X mutants and wild type mice illustrate indistinguishable changes in muscularized vessels after administration of saracatinib and dasatinib. Images are taken at 10x.