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INCB054329 reduces the host response to SARS-CoV-2 infection in K18-hACE2 mouse hearts

We next assessed the response to SARS-CoV-2 infection in vivo.

Because mice are not susceptible to SARS-CoV-2 infection, we used a recently described K18-hACE2 model (Oladunni et al., 2020) to study the response and effects of BETi (Figure 5A).

SARS-CoV-2-infected mice had severe lung pathology and substantial viral RNA reads in the lungs at 4–5 days post infection, confirming successful lung infection (Figures 5B and 5C). RNA-

seq of the lungs revealed increased expression of 419 genes (Figure 5D) strongly associated with a viral response (Figure 5E; Table S1A). 

Concordant with our hCO data, Stat1 and Ep300 were the top predicted transcriptional regulators (Figure 5F; Ta-

ble S1B). Interestingly, there was only negligible infection of the heart (Figure 5C) and no obvious pathology including necrosis, immune cell infiltration, or fibrosis (Figure 5G). 

However, there was a substantial and robust upregulation of a viral response in the heart with ECM repression observed (Figures 5H and 5I; Table S1A) including Col1a1, Col3a1, and Col4a2. This was again enriched for Stat1 and Ep300 as top predicted transcriptional regulators (Figure 5J; Table S1B), indicating a robust systemic response in the hearts of SARS-CoV-2-infected mice. 

This response could be partially blocked by INCB054329 treatment with repression of 91 genes that were enriched for the viral response (Figures 5K–5M; Table S1A). 

This response was more specific to the heart, because INCB054329 did not regulate any genes in the lungs (data not shown). The repression by INCB054329 was predicted to be primarily mediated via Ep300 rather than Stat1 (Figure 5N; Table S1B). These results werefurther supported by ingenuity pathway analysis of upstream regulators revealing strong activation signatures for IFNG, poly rI:rC-RNA, and Stat1 in both lungs and hearts of K18-hACE2 SARS-CoV-2-infected mice, which INCB054329 strongly inhibited (Table S1C). Potentially important mediators and markers of the response

were found by integrating the multiple datasets. The CS induced response in the hCOs (either fibroblasts or cardio-

myocytes) and hearts of SARS-CoV-2-infected K18-hACE2

mice shared 32 regulated genes (Figure 5O) that were en- riched for the viral response (Figure 5P). The consistent transcriptional program in CS-treated hCOs (both fibroblasts and cardiomyocytes) and SARS-CoV-2-infected K18-hACE2

mice, which were also downregulated genes by INCB054329 treatment in vivo, revealed 5 key targets. These comprise the key inflammatory genes Nmi, Tap1, B2m, Stat1, and Lgals3bp (Figure 5Q). 

Of particular interest is LGALS3BP (galectin-3 binding protein), because it has been shown to be a top-pre-

dictor of COVID-19 severity in humans (Messner et al., 2020) and we therefore interrogated its expression in our subsequent models.

INCB054329 protects against inflammatory mediated

dysfunction in vivo and by COVID-19 serum

The study into the efficacy of SARS-CoV-2-related cytokine

storm therapeutics on the heart in vivo is technically challenging in biosafety level 3 and because the severe lung/brain infection in K18-hACE2 mice causes a rapid decrease in weight and requires

euthanasia (Figure 5R). We therefore used surrogate models.

We used a LPS-induced cytokine storm mouse model (Figure 6A). LPS induced pro-inflammatory cytokines TNF, IL-1b,

and IFN-g, which were elevated in the plasma (Figure 6B), along with Lgals3bp in the heart (Figure 6C). Treatment with

INCB054329 blocked the LPS-induced pro-inflammatory cytokine production (Figure 6B) and Lgals3bp induction in the heart (Figure 6C). 

We observed a marked improvement in mortality, whereby all INCB054329-treated mice survived after 24 h of the LPS-challenge, compared with only 25% in the control group

(Figure 6D). 

To determine whether BETi could treat an established LPS-induced cytokine storm, we delayed injection of

INCB054329 1.5 h after LPS injection and assessed cardiac function at 6 h (Figure 6E). 

INCB054329 fully prevented the decrease in cardiac function observed after LPS injection (Fig-

ure 6F). 

Together, these findings demonstrate that BETi using INCB054329 has robust effects in preventing inflammatory-

induced cardiac dysfunction in vivo.

The factors present in COVID-19 patient serum are more complex than our CS conditions. We assessed the impact of this serum on hCOs (Figure 6G; Tables S2A and S2B). Our most potent CS factor, IFN-g, correlates with COVID-19 disease progression and is elevated in patient serum in one of the most

comprehensive profiling studies to date (Ren et al., 2021). We found that IFN-g was higher in patients with elevated BNP as a marker of cardiac stress (>0.3 ng/mL) but not CTNI as a marker

of acute injury (>0.5 ng/mL) (Figures 6H and 6I). Factors in human serum can alter hCO function, because patients receiving noradrenaline as inotropic support had elevated contractile force

in hCOs (Figure 6J). Diastolic dysfunction was induced in

hCOs by serum with elevated BNP (Figure 6J) with no viral infec-

tion detected (data not shown), and INCB054329 could prevent this response (Figure 6K). We also found that LGALS3BP induction could be prevented by treatment with multiple BETi

(Figure 6L).

Collectively, these data indicate that BET inhibition with INCB054329 prevents cardiac dysfunction in multiple complex

inflammatory models, as well as repressing the key COVID-19

severity marker LGALS3BP.

INCB054329 decreases hACE2 expression and reduces

SARS-CoV-2 in hPSC-cardiac cells

SARS-CoV-2 potentially infects human hearts and has been shown to infect human pluripotent stem cell-derived cardiac cells (hPSC-CM) (Sharma et al., 2020). Recently, other investiga-

tors have also demonstrated that BETi reduced Ace2 in vivo and SARS-CoV-2 infection (Qiao et al., 2020). We sought to determine whether BETi blocked infection (Figure S7A).

We confirmed previous findings using 2D cultured hPSC-cardiac cell infection studies, where increasing the MOI increased the degree of cell death (Figure S7B). Infection with a low MOI

(0.01) was sufficient for viral replication and cell death over 7 days (Figure S7C). A 3-day pre-incubation of INCB054329

was sufficient to reduce ACE2 expression ~4-fold (Figure S7D).

Consequently, pre-treatment with INCB054329 reduced SARS-CoV-2 N protein expression (Figure S7E) and intracellular viral RNA (Figure S7F). In addition to INCB054329, the widely

used BETi JQ-1 reduced SARS-CoV-2 RNA (Figure S7G). In our SARS-CoV-2 K-18 mouse infection studies, INCB054329 treatment reduced endogenous Ace2 in hearts in vivo (Fig-

ure S7H). Thus, BETi also has potential to block SARS-CoV-2 infection of cardiac cells in addition to preventing dysfunction. 

BETi for translation to the clinic

We assessed the ability of all commercially available BETi com-

pounds to prevent CS-induced diastolic dysfunction in hCOs. We found that all compounds prevented dysfunction except for ABBV-744 (Figure 7A). BETi with dual bromodomain 1 (BD1) and bromodomain 2 (BD2) activities display side effects (Gilan et al., 2020); as such, it is critical that we determine the

bromodomain selectivity of the response. 

BD2-selective drugs, such as ABBV-744 and apabetalone, have limited side effects.

Apabetalone has been used for up to 3 years in >1,700 humans at risk of cardiac disease, with efficacy in preventing heart failure and a favorable safety profile (Nicholls et al., 2021; Ray et al.,

2020). 

ABBV-744 elevated diastolic dysfunction (Figure 7A), and we suspect its lack of efficacy was due to its on-target inhibition of the androgen receptor (AR). 

BD2-specific efficacy was confirmed using a BD2-specific molecule, RXV-2157 (Figure 7B), and we also confirmed efficacy with the BD-2 selective apabet-

alone (Figures 7A and 7B). 

Additionally, analysis of plasma from

the ASSURE phase IIb clinical trial indicated that BD2-selective

apabetalone reduced LGALS3BP in patients with cardiovascular

disease (Figure 7C), a marker of COVID-19 severity (Messner

et al., 2020).

We confirmed the BD2-specificity of blocking SARS-CoV-2 infection. The BD2-specific RXV-2157 and BD2-selective apabetalone molecules downregulated hACE2 (Figure 7D), which led to decreased surface expression (Figure S7I) and SARS-CoV-2 spike protein binding (Figure S7J). 

These compounds also reduced SARS-CoV-2 loading (Figures 7E, 7F, and S7K) and viral titer, including a 2.6-fold decrease in viral titer with apa-

betalone (Figure 7G).

Together, this demonstrates that BD2-selective BETi drugs are lead candidates for rapid clinical translation to prevent COVID-19 injury in the heart.

DISCUSSION

We define CS conditions resulting in severe diastolic dysfunction with 20%–50% increases in time to 50% relaxation in hCOs. This is consistent with ~13%–18% increases in cardiomyocytes derived from patients with heart failure with preserved ejection

fraction (HFpEF), with similar absolute increases of 100–150 ms (Runte et al., 2017). It is also consistent with diastolic

dysfunction reported in COVID-19 patients (Szekely et al., 2020), indicating our hCO model recapitulates key clinical features of diastolic dysfunction.

Our transcriptional profiling revealed a striking similarity between the inflammatory response in CS-treated hCOs and hearts of SARS-CoV-2-infected K18-hACE2 mice. CS also elicited a

more pronounced transcriptional response in the fibroblasts

within hCOs (Figures 3C and 3D). This was with negligible viral infection in our models, meaning that inter-organ and intra-organ signaling appears to play a key role in inflammation-induced car-

diac dysfunction. It will be important to decipher the systemic and intra-organ drivers of dysfunction in other organs, as

COVID-19 and many other inflammatory diseases can result in multi-organ dysfunction.

Our data establish BET inhibition as a viable therapeutic strategy to attenuate cytokine storm-induced cardiac dysfunc-

tion. Previously, BETi compounds have shown efficacy in small animal cytokine storm models (Nicodeme et al., 2010). There is also compelling evidence implicating bromodomain proteins as

key mediators in pathological pro-fibrotic signaling in heart failure (Duan et al., 2017; Stratton et al., 2019), in experimental models of pressure overload and myocardial infarction-induced HF (Anand et al., 2013), and in genetic cardiomyopathies

(Antolic et al., 2020; Auguste et al., 2020). However, this study is instrumental in establishing BET inhibition as a therapeutic intervention to prevent cardiac dysfunction caused by

inflammation.

Clinical data from COVID-19 patients also point to additional cardiac pathologies. Microthrombi were reported in the hearts of 14 out of 40 patients (35%) that died from COVID-19, which

was associated with areas of myocardial necrosis (Pellegrini et al., 2021). Consistent with these observations, we observed that ‘‘complement and coagulation cascades’’ were enriched

in the lungs of K18-hACE2 mice with SARS-CoV-2 infection (Figure 5E; Table S1) and were likely related to the viral-induced inflammatory response. Serpine 1 is a key inhibitor of tissue-type

plasminogen activator and urokinase-type plasminogen activator and is required for fibrinolysis downregulation and degradation of blood clots. Concordantly, serpine1 (also known asplasminogen activator inhibitor 1), was upregulated in both the lungs (3.9-fold) and in the heart (2.8-fold, both FDR <0.05) of SARS-CoV-2-infected K18-hACE2 mice, which was indeed

abrogated in the heart upon treatment with the BETi INCB054329 (FDR <0.05). 

In addition, arrhythmic events have

been widely reported in COVID-19 patients (Nishiga et al., 2020). Indeed, we observed that arrhythmic events increased in hCOs with CS, for which INCB054329 also conferred protec-

tion (Figures S5L–S5N). These data suggest that BET inhibition may be effective in attenuating multiple deleterious aspects of systemic inflammation on the heart that warrant further investigation.

We demonstrated that BETi are attractive therapeutic candidates, however, the side effect profiles of some BETi may preclude their use in the clinic. Genetic ablation studies have shown that BRD4 plays an integral homeostatic role in cardiomyocytes, suggesting that the loss of BET proteins may have detrimental

effects on mitochondrial energy production (Kim et al., 2020;

Padmanabhan et al., 2020). 

Emerging evidence dissecting the

roles of BD1 and BD2 bromodomains in inflammatory disease models has indicated that BD2-selective inhibition preferentially blocks the induction of gene expression while minimally affecting established transcription programs (Gilan et al., 2020). More

recently, BD2-selective drugs such as ABBV-744 and apabetalone have been developed to overcome these side-effect profiles. 

Although ABBV-744 was not effective in our hCO model (potentially due to its targeting of AR), we demonstrate that

BD-2 selective compounds RXV-2157 and apabetalone demonstrate efficacy. 

This underscores the need for careful BETi selection, despite broad ability to modulate critical target genes (Fig-

ure 6L) and utility for a variety of clinical conditions (Cochranet al., 2019). 

Importantly, BD2-selective BETi apabetalone reduced CS-induced diastolic dysfunction and downregulated

ACE2 and reduced viral infection (Figure 7). 

Taken together, the efficacy and known safety profile of apabetalone make it a

prime candidate to protect against cardiac injury for inflammatory diseases such as COVID-19.

The overlap in risk factors for HFpEF and COVID-19 mortality suggests that our findings may also have broader implications. HFpEF risk factors including diabetes and obesity are also associated with chronic inflammation. Recent studies have shown

that elevated inflammatory markers are associated with worsening heart function in HFpEF (Sanders-van Wijk et al., 2020), thus indicating that inflammation may drive dysfunction across multiple cardiac diseases, and BET inhibitors are putative thera-

peutic candidates.

Limitations of study

Human COVID-19 patient serum and CS directly impacted hCO function. There is evidence that the heart can be inflamed in patients with COVID-19 (Kotecha et al., 2021; Puntmann et al., 2020), but whether direct cardiac inflammation is required for functional impact or whether the systemic environment is a driver of cardiac dysfunction remains to be determined. Larger studies are required to ascertain the full extent of the inflammatory effects on heart function in the clinic, in particular on diastolic function as found in some echocardiography studies

(Szekely et al., 2020). It will be important to determine whether cardiac inflammation and its functional effects (that may be sub-clinical in some cases) are prolonged following acute infec-

tion and whether this predisposes patients to future risk of cardiovascular events.

Our hCO model is free from an active immune system and the secondary effects of neurohormonal compensation present in vivo. Our hCO work, the lack of response in the lungs of INCB054329-treated SARS-CoV-2-infected K18-hACE2 mice, and improvement of heart function with delayed INCB054329 treatment in the LPS-treated mice (Figure 6F) all indicates robust cardiac-specific effects. However, BETi within an in vivo setting may also reduce immune responses and cytokine induction (e.g., Figure 6B), thus, we cannot rule this out as a potential mechanism for cardiac protection in mouse studies. In order to elucidate cardiac-specific effects, genetic studies could be useful. However, these may be difficult because (1) BRD4 knockout has different effects to small molecule inhibitors (Kim et al.,2020; Padmanabhan et al., 2020), and (2) BETi drugs bind multiple BRD family members, which may be more potent that targeting one member (Gilan et al., 2020). Further mechanistic insight into the key transcriptional targets of BET proteins is important. These targets could then be manipulated using cardiac-specific genetic approaches to validate cardiac specificity of BETi. However, the feasibility of this approach will be dependent on the number of targets that are critical for BETi efficacy, as multiple targets may require simultaneous genetic manipulation.