(R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages

Cystic fibrosis (CF) is characterized by chronic bacterial infections and heightened inflammation. Widespread ineffective antibiotic use has led to increased isolation of drug resistant bacterial strains from respiratory samples. (R)-roscovitine (Seliciclib) is a unique drug that has many benefits in CF studies. We sought to determine roscovitine’s impact on macrophage function and killing of multi-drug resistant bacteria. Human blood monocytes were isolated from CF (F508del/F508del) and non-CF persons and derived into macrophages (MDMs). MDMs were infected with CF clinical isolates of B. cenocepacia and P. aeruginosa. MDMs were treated with (R)-roscovitine or its main hepatic metabolite (M3). Macrophage responses to infection and subsequent treatment were determined. (R)-roscovitine and M3 significantly increased killing of B. cenocepacia and P. aeruginosa in CF MDMs in a dose-dependent manner. (R)-roscovitine-mediated effects were partially dependent on CFTR and the TRPC6 channel. (R)-roscovitine-mediated killing of B. cenocepacia was enhanced by combination with the CFTR modulator tezacaftor/ivacaftor and/or the alternative CFTR modulator cysteamine. (R)-roscovitine also increased MDM CFTR function compared to tezacaftor/ivacaftor treatment alone. (R)-roscovitine increases CF macrophage-mediated killing of antibiotic-resistant bacteria. (R)-roscovitine also enhances other macrophage functions including CFTR-mediated ion efflux. Effects of (R)-roscovitine are greatest when combined with CFTR modulators or cysteamine, justifying further clinical testing of (R)-roscovitine or optimized derivatives.

Scientific Reports | (2020) 10:21700 | https://doi.org/10.1038/s41598-020-78817-x www.nature.com/scientificreports/ (R)-roscovitine (Seliciclib, hereafter refered to as roscovitine) is a synthetic, low molecular weight, tri-substituted purine (Fig. 1), initially developed as a cyclin-dependent kinase inhibitor for the treatment of lung, breast, and nasopharyngeal cancers [8][9][10][11] . It has undergone many preclinical studies as well as phase 1 and phase 2 clinical trials in more than 520 patients, mostly as a potential anti-cancer drug. Roscovitine synthesis is a cheap, fast, optimized 3-step protocol 12 . The drug has good oral and lung availability, a short half-life, and the main hepatic metabolite (M3, Fig. 1) is inactive on kinases, but displays some other biological activities. Roscovitine has shown multiple benefits in CF studies [13][14][15] . Despite its lack of kinase inhibitory activity, the M3 metabolite of roscovitine is able, like roscovitine, to correct the intraphagolysosomal pH of alveolar macrophages and enhance their bactericidal activity 14 , and correct F508del-CFTR trafficking 15 . Recently, roscovitine was tested in the CF population in a Phase 2A trial 16 (ROSCO-CF: A Phase II, dose ranging, multicenter, double-blind, placebo controlled study to evaluate safety and effects of [R]-roscovitine in adults subjects with CF, with at least one F508del-CFTR mutation and chronically infected with Pseudomonas aeruginosa). We sought to build upon this trial and further determine the efficacy of roscovitine in CF, through examination of its impact upon restoration of macrophage function, including the ability to kill multi-drug resistant Burkholderia cenocepacia.

Methods
Subjects. People with a confirmed CF diagnosis and homozygous for the F508del mutation were recruited from the outpatient CF clinic at baseline health. People were excluded if using chronic immunosuppressants or if they had a history of transplantation. The diagnosis of CF was defined as two disease-causing mutations or a sweat chloride test ≥ 60 mmol/L. Human subject recruitment was approved by the Institutional Review Board of Nationwide Children's Hospital (IRB15-00611). Informed consent and/or assent was obtained for all participants. All methods were performed in accordance with the Declaration of Helsinki for medical research involving human subjects. A parent or guardian of child participants provided informed consent on their behalf. Healthy, age and gender-matched controls were also recruited.
Reagents. Roscovitine and M3 were synthesized at ManRos Therapeutics and used at concentrations ranging from 0.1 to 100 μM. TRPC6 inhibitor 17 was provided by Dr. Stephanie Häfner and used at 10 µM. CFTR modulators tezacaftor and ivacaftor (Selleck chem) were used at 5 µM. Cysteamine (Sigma) was used at 5 µg/ml and the CFTR inhibitor Inh-172 (Sigma) was used at 5-10 µM. All reagents were dissolved in DMSO and therefore non-treatment conditions received equal DMSO concentrations. All reagents were given as pre-treatment for 6 h prior to general infection experiments, and 4 h prior to halide efflux and phagocytosis.
Statistics. Statistical analyses were performed using GraphPad Prism software (version 7.05). Two sample unpaired t-tests were used for independent sample comparisons. One-way ANOVA with post-hoc Tukey tests were used for multiple comparisons. Statistical significance was defined as a p value < 0.05.

Conference presentation. Portions of this manuscript were presented at the 2019 North American Cystic
Fibrosis Conference.

Results
Patient demographics. The demographic characteristics of the persons who donated blood for the study are listed in Table 1. The CF donors were relatively homogenous, with a targeted Phe508del homozygous population to avoid genotype confounders. Of note, approximately 40% of CF patients were on a CFTR modulator at blood draw (lumacaftor/ivacaftor or tezacaftor/ivacaftor). This was consistent with our overall clinic population during the study timeframe.

Roscovitine increases CF macrophage-mediated killing of multi-drug resistant bacteria.
Roscovitine and M3, its main hepatic carboxylated metabolite 25,26 , are low molecular weight, 2,6,9-trisubstituted purines which match the Lipinski "rule of five" which defines molecules with a high drugability potential ( Fig. 1) 27 . To determine the impact of roscovitine on macrophage-mediated killing of B. cenocepacia, we isolated MDMs from CF and non-CF donors and infected them with B. cenocepacia prior to treatment with increasing concentrations of roscovitine or M3. Both roscovitine ( Fig. 2A) and M3 (Fig. 2B) demonstrated a significant reduction in bacterial load in a dose-dependent manner, with greater than one log reduction in bacterial load observed at 50 μM. Concentrations beyond 50 μM were associated with increased cell death (data not shown). Both roscovitine and M3 also reduced B. cenocepacia burden in non-CF MDMs ( Fig. 2A,B). We then examined Scientific Reports | (2020) 10:21700 | https://doi.org/10.1038/s41598-020-78817-x www.nature.com/scientificreports/ roscovitine and M3 compared to and in combination with the compound cysteamine, as we recently demonstrated that cysteamine increased macrophage-mediated killing of common CF pathogens 22 . Cysteamine alone demonstrated a similar reduction in B. cenocepacia load in CF MDMs compared to 50 μM roscovitine ( Fig. 2A) and M3 (Fig. 2B). However, there was an increased dose-dependent reduction in bacterial load in CF MDMs when cysteamine was combined with increasing concentrations of roscovitine ( Fig. 2A) or M3 (Fig. 2B). When cysteamine was combined with 50 μM roscovitine or M3 there was a 4 to 5 log reduction in bacterial load. A similar trend was observed in non-CF MDMs.   www.nature.com/scientificreports/ Next, we examined the capacity of CF MDMs treated with roscovitine or M3 to kill a multi-drug resistant clinical strain of P. aeruginosa isolated from a clinic patient. Roscovitine (Fig. 2C) and M3 (Fig. 2D) both demonstrated a dose-dependent reduction in P. aeruginosa bacterial load in CF MDMs, similar to B. cenocepacia. However, there was no change in bacterial load in non-CF MDMs for roscovitine, but a significant decrease with M3. In contrast to B. cenocepacia, treatment of P. aeruginosa-infected MDMs with cysteamine alone was equivalent to 5-20 μM concentrations of roscovitine (Fig. 2C) or M3 (Fig. 2D). However, we did observe an increased effect of cysteamine on P. aeruginosa bacterial load when combined with roscovitine (Fig. 2C) or M3 (Fig. 2D) in both CF and non-CF MDMs.
Because of the observed effects of roscovitine and M3 on macrophage-mediated bacterial killing, we tested the ability of roscovitine and M3 to kill B. cenocepacia in the absence of macrophages. In a direct killing assay, we saw a similar impact of roscovitine and M3 upon B. cenocepacia bacterial load, with a dose-dependent decrease in bacterial load with increasing concentrations up to 100 μM (Fig. 3A). A direct reduction in bacterial load was not achieved until concentrations of 50 μM (Fig. 3A).
Roscovitine's impact on killing of bacteria is partially dependent on CFTR and TRPC6. Roscovitine has broad mechanistic actions, including stabilization of CFTR and indirect activation of the TRPC6 Ca 2+ channel 14,15 . To further test the role of roscovitine when CFTR is present but functionally inhibited, we isolated non-CF MDMs and infected them with B. cenocepacia in the presence or absence of a CFTR inhibitor and increasing concentrations of roscovitine. CFTR inhibition alone increased B. cenocepacia bacterial load compared to uninhibited non-CF MDMs (Fig. 3B). Treatment with 50 μM roscovitine significantly reduced bacterial load in non-CF MDMs (Fig. 3B). Roscovitine treatment of CFTR inhibited MDMs also recapitulated our www.nature.com/scientificreports/ prior finding of reduced bacterial load in primary CF MDMs (Fig. 3B). A significant difference in bacterial load between non-CF MDMs and CFTR-inhibited MDMs persisted during roscovitine treatment (Fig. 3B). These results suggest a partial dependence on CFTR for roscovitine-mediated bacterial killing.
To test the role of the TRPC6 channel in CF MDMs during roscovitine treatment, non-CF and primary CF MDMs were infected with B. cenocepacia in the presence or absence of a TRPC6 inhibitor 17 (10 µM, 4 h) and/ or roscovitine or M3. TRPC6 channel inhibition alone did not directly impact bacterial load in either CF or non-CF MDMs (Fig. 3C). However, there was a significant reduction in bacterial load for both CF and non-CF MDMs when TRPC6-inhibited MDMs were treated with either roscovitine or M3 (Fig. 3C). The reduced bacterial load during roscovitine/M3 treatment and TRPC6 inhibition was attenuated in magnitude when compared to Fig. 2 results for roscovitine or M3 alone. These results suggest that roscovitine-mediated bacterial killing is partially dependent on TRPC6.

Roscovitine enhances CFTR modulator-induced functional changes in macrophages.
We recently demonstrated that CF MDMs have defective bacterial phagocytosis, which can be partially rescued by administration of a CFTR modulator 20 . To determine if roscovitine or M3 enhanced killing of bacteria via improved phagocytosis, we measured phagocytosis of B. cenocepacia in CF MDMs in the presence or absence of roscovitine, M3, or the CFTR modulator tezacaftor/ivacaftor. Untreated CF MDMs demonstrated a significant reduction in phagocytosis at baseline compared to non-CF (Fig. 3D), recapitulating our prior findings 20 . There was no improvement in CF phagocytosis with the addition of tezacaftor/ivacaftor, roscovitine, or M3 alone (Fig. 3D). There was no significant improvement in phagocytosis when tezacaftor/ivacaftor was combined with either roscovitine or M3 (Fig. 3D). These results suggest that roscovitine does not exert its primary effects through enhanced bacterial phagocytosis.
Next, we measured changes in CFTR-dependent chloride efflux in CF and non-CF MDMs as a surrogate of CFTR function. MDMs were infected with B. cenocepacia in the presence or absence of tezacaftor/ivacaftor, roscovitine, M3 or combinations. Using a halide efflux assay, we found that CF MDMs had significantly reduced CFTR function at baseline compared to non-CF MDMs (Fig. 4A). Infection with B. cenocepacia significantly reduced CFTR function in non-CF MDMs and further reduced CFTR function in CF MDMs (Fig. 4A). Treatment of B. cenocepacia-infected CF MDMs with tezacaftor/ivacaftor or roscovitine alone led to increases in CFTR function up to the level observed in infected non-CF MDMs. Only a combination of tezacaftor/ivacaftor and roscovitine significantly increased CFTR function in CF MDMs during infection compared to no treatment (Fig. 4A). In contrast to roscovitine plus tezacaftor/ivacaftor, the combination of M3 and tezacaftor/ivacaftor did not change CFTR function compared to tezacaftor/ivacaftor alone (Fig. 4A).
To determine if CFTR expression was also impacted by infections or treatments, we measured CFTR protein expression during infection with B. cenocepacia in the presence or absence of tezacaftor/ivacaftor, roscovitine, M3, or combinations. We found that infection alone was not associated with changes in CFTR protein expression by western blot in CF or non-CF MDMs (Fig. 4B,C). Treatment with roscovitine alone, roscovitine plus tezacaftor/ivacaftor, M3 alone or M3 plus tezacaftor/ivacaftor all were associated with a trend towards increased CFTR expression in CF MDMs during infection, but variability was noted within donors (Fig. 4B,C). Interestingly, treatment with M3 decreased CFTR expression in some non-CF donors and the combination of roscovitine plus tezacaftor/ivacaftor significantly increased CFTR expression in non-CF (Fig. 4B,C). Examples of uncropped western blots for Fig. 4 can be found in Supplemental Fig. 1.
We then performed a CFU assay in CF MDMs during infection with B. cenocepacia in the presence of tezacaftor/ivacaftor with or without roscovitine or M3. CF MDMs treated with tezacaftor/ivacaftor alone had a significant reduction in bacterial burden (Fig. 4D). However, the combination of roscovitine and tezacaftor/ ivacaftor further reduced bacterial burden compared to tezacaftor/ivacaftor alone (Fig. 4D). Similar to our CFTR functional results, the combination of M3 and tezacaftor/ivacaftor did not change bacterial burden compared to tezacaftor/ivacaftor alone (Fig. 4D).
Last, because we had previously shown that cysteamine combined with roscovitine had additive effects on killing of B. cenocepacia, we performed a CFU assay for combinations of roscovitine/M3, tezacaftor/ivacaftor, and cysteamine. Cysteamine treatment combined with tezacaftor/ivacaftor significantly reduced bacterial burden compared to infection alone (Fig. 4E). The addition of roscovitine but not M3 to cysteamine/tezacaftor/ivacaftor led to a further reduction in bacterial burden by at least 1 log compared to the cysteamine/tezacaftor/ivacaftor combination alone (Fig. 4E).
Combined, our data suggest efficacy of roscovitine-mediated killing of resistant CF pathogens, but the greatest efficacy is achieved when a multi-pronged approach is utilized with CFTR modulators and cysteamine.

Discussion
Treatment of multi-drug resistant bacterial infections remains an important clinical problem in many diseases, including CF. In particular, Burkholderia infections in CF are extremely difficult to treat, lacking standardized antibiotic regimens 28 , an exclusion to lung transplantation and often from clinical trials testing new anti-bacterial products. Furthermore, there is a growing incidence of Burkholderia cepacia complex healthcare-acquired infections in patients without CF 29,30 . To this end, we demonstrated the ability of roscovitine to enhance macrophagemediated killing of Burkholderia cenocepacia as well as multi-drug resistant Pseudomonas aeruginosa. Roscovitine also showed enhanced efficacy in combination with other existing therapeutics such as CFTR modulators and cysteamine. Our findings have important implications for future therapeutic regimens in CF.
Although we demonstrated efficacy of roscovitine alone or in combination with the CFTR modulator tezacaftor/ivacaftor, the strongest effect of roscovitine was in combination with the transglutaminase 2 inhibitor cysteamine. Previously we demonstrated that cysteamine reduces transglutaminase 2 accumulation in CF 22 , Scientific Reports | (2020) 10:21700 | https://doi.org/10.1038/s41598-020-78817-x www.nature.com/scientificreports/ leading to increased Burkholderia uptake into autophagosomes and subsequent improved clearance. In our current study we showed that roscovitine has a similar increase in killing of B. cenocepacia compared to cysteamine, but combined they reduce bacterial burden by 4-5 log. In contrast, we found more incremental additive effects of roscovitine and cysteamine when macrophages were infected with P. aeruginosa. However, CF macrophages were more easily able to control P. aeruginosa burden, with lower starting and treatment-related bacterial burden compared to B. cenocepacia. These results suggest that pathogen specific effects (such as subversion of autophagy for B. cenocepacia) may be important in dictating which bacteria require a combinatorial treatment approach, compared to solitary treatment with roscovitine. Further, we do not know if the observed reductions in B. cenocepacia with roscovitine treatment alone would be clinically significant.
In addition to an impact upon killing of bacteria, we found that roscovitine increases CFTR function in primary CF macrophages, particularly when combined with the CFTR modulator tezacaftor/ivacaftor. Roscovitine has known effects on CFTR, having been shown to inhibit CFTR degradation by proteases and subsequently lead to increased CFTR trafficking to the plasma membrane and partial restoration of CFTR function 15 . Roscovitinemediated increases in macrophage CFTR function mirrored changes in killing of B. cenocepacia, with a greater reduction in bacterial burden when roscovitine was combined with tezacaftor/ivacaftor compared to tezacaftor/ ivacaftor alone. Our results further support the concept that roscovitine could have pleiotropic effects in CF through restoration of CFTR function. Further work will be needed to determine how roscovitine interacts with emerging CFTR modulator combinations such as tezacaftor/ivacaftor/elexacaftor 31 , which have shown improved clinical benefits compared to tezacaftor/ivacaftor. Despite a relatively good tolerance, roscovitine did not demonstrate efficacy in the ROSCO-CF clinical trial (Meijer et al., unpublished). There are many possible reasons that could explain the discrepancy between the encouraging results obtained with roscovitine on various cell 14,15 and animal (Bonfield T et al., unpublished) models of CF and the disappointing lack of beneficial effects of roscovitine in the clinic. Some of these reasons may be the short duration of the trial, the low number and biological diversity of patients, and inappropriate dosing (dose, formulation, administration mode, frequency of administration and timing [circadian rhythm www.nature.com/scientificreports/ was not taken into account]). Another reason may stem from the fact that roscovitine was not optimized for CF, and just re-purposed from anticancer activity to the CF pathology. Classical medicinal chemistry guided optimization starting from roscovitine and M3 should lead to a drug candidate with much improved efficacy. In addition, this optimization should be carried out in a combination approach with established CFTR modulators, as clearly encouraged by the results presented here. Finally, it is possible that the strong anti-inflammatory effects or roscovitine 13 could have a negative impact on host responses to chronic infection in CF, and negate potential benefits mediated by enhanced killing of bacteria. We believe that this possibility is less likely given roscovitine's overall safety profile, success in CF pre-clinical studies, and benefits of other anti-inflammatories in CF 16 such as azithromycin or ibuprofen. Never-the-less, changes in inflammatory responses during future trials of roscovitine or metabolites are an important design consideration.
In summary, our data support the use of roscovitine or derivatives of it for multi-drug resistant bacterial infections in CF. Roscovitine enhances macrophage function including CFTR-mediated ion efflux and has additive benefits when combined with existing CFTR modulators or cysteamine. Further clinical trial testing of roscovitine and optimized derivatives and analogues in CF is warranted.

Data availability
Data are available upon request to the authors.