Cell culture and compounds

All cell lines were cultured at 37°C in a humidified incubator with 5% CO2. Cell culture media and supplements were obtained from Thermo Fisher Scientific. All cell lines were obtained from ATCC (American Type Culture Collection, USA) unless stated otherwise and cultured according to instructions. JBI-097, Iadademstat and Ricolinostat were synthesised in-house by the Jubilant Drug Discovery Unit of Jubilant Biosys Ltd. The various batches of JBI-097 utilized in these studies consistently exceeded a purity of 97% as measured by Nuclear Magnetic Resonance (NMR), Liquid Chromatography- Mass Spectroscopy (LC-MS) and elemental analysis. All compounds were dissolved in dimethylsulphoxide (DMSO) as 10 milli molar stock solutions and stored -20°C. Pomalidomide and Bortezomib were purchased from selleckchem.

Mice

All in vivo experiments were carried out at Jubilant Biosys Ltd., which has AAALAC (Association for Assessment and Accreditation of Lab Animal Care International) accreditation. Animals were quarantined for a week upon arrival and healthy animals were approved for the study.

All SCID beige and Balb/c mouse used in this study were purchased from Vivo Biotech in Hyderabad, India [under licence from Taconic]. The animals were housed in techniplast individually ventilated cages (n = 5 per cage) in a temperature-controlled environment with a 12-hour light/dark cycle. Animals were fed enough with an autoclaved commercial diet (Nutrilab Rodent Feed, spherical pellets) and autoclaved water ad libitum. All the animals were monitored daily by the same staff for the duration of the experiment, and were weighed three times per week. The animal studies associated with Fig 5a-5f were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IAEC/JDC/2019-174R). During the study, the care and use of animals was in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals, 8^th Edition, 2010 (National Research Council). Institutional Animal care and Use Committee (IACUC) approved general humane endpoints for tumor models were applied to this study. Mice were euthanized at the completion of the study protocol by carbon dioxide overdose. Mice were sacrificed following the development of any of the following clinical signs of tumor progression: severe head tilting, ataxia, circling, lethargy and/or weight loss ≥20% of the initial weight.

LSD1 expression and inhibition assay

Human recombinant LSD1 was expressed and purified as described below. The N-terminal truncated Human LSD1 (Isoform 1): 172a.a-852a.a containing SWIRM Domain was PCR amplified from HEK293 cDNA, sub‐cloned into a pFastbac1 vector, and expressed in insect cells carried out using Bac-to-Bac system with cleavable N-terminal 6xHis tag. Briefly, Sf9 cells in suspension culture were full-grown at 27°C in 2.8 liters baffled flasks containing a 1000 ml of SF900 II SFM medium (Gibco), shaken at 70 rpm. The cells were infected at a density of 1.2–1.5 x 10⁶ cells/mL using a multiplicity of infection (MOI) of 2. One mL samples were collected at indicated times to measure viability (Cedex Cell counter), and expression was evaluated by western blot using an antibody against the 6 His tag. 12g of cell pellet, was harvested from 2 L of culture, re-suspended (Resuspension/Binding Buffer: 20 mM HEPES pH 7.9, 300 mM NaCl, 0.5 mM TCEP, 10% Glycerol, 1X Protease inhibitor cocktail), sonicated, clarified by centrifugation. Then, the supernatant was loaded onto 5 mL Ni-affinity chromatography resin (GE Healthcare), the column was washed with the 10 column volumes (CV) of binding buffer supplemented with increasing concentrations of imidazole (5 to 35 mM). Imidazole and protein eluted in 10 CV gradient with 35–500 mM Imidazole. Then, the protein was further purified by Superdex-200 Size exclusion chromatography (GE Healthcare) (20 mM HEPES pH 7.9, 150 mM NaCl, 1.0 mM TCEP, 10% Glycerol) and the purified enzyme was used for the assay. LSD1 inhibitor tranylcypromine (TCP), was procured from Selleckchem. LSD1 enzyme, TCP, and biotinylated peptide substrate were diluted in assay buffer just before use. 2X inhibitor (10 μL, diluted in assay buffer) or assay buffer, and 5 nM enzyme were added to a 96-well plate and incubated at room temperature for 30 min. 5 μL of biotinylated histone H3K4me1 peptide (4X) was added to each well and incubated at room temperature (RT) for 1 h. Stop solution containing 300 μM TCP in 1X LANCE detection buffer from Perkin Elmer was added to the wells and incubated for 5 min at RT. Then, a detection mix containing 2 nM Eu-Ab and 50 nM ULight-Streptavidin (Perkin Elmer) in 1X LANCE detection buffer was prepared and added to the reaction mixture. This mixture was incubated for 1 hour at room temperature. Readings were taken using the Pherastar Reader in TR-FRET mode (excitation at 337 nm & emission at A-665 nm, B-620 nM). All chemicals were purchased from Sigma–Aldrich unless specified.

HDAC isoform inhibition assay

Dose-response studies for HDAC isoforms were conducted at Reaction Biology Corporation. All assays are based on the same principle of a two-step reaction: first, the substrate with the acetylated lysine side chain is incubated with a sample containing HDAC enzyme, to produce the deacetylated products, which are then digested in the second step by the addition of the developer to produce the fluorescent signal proportional to the amount of deacetylated substrates. Recombinant proteins HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and HDAC10 were incubated with a test compound for 5min, and then the fluorophore-conjugated substrate was added to the wells and incubated for 30 min to 2 h for different HDACs. Reactions were performed in assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA, 1% DMSO, pH 7.4) and fluorescence measurements were obtained approximately every 20 min for an hour using an Envision, Perkin-Elmer plate reader (Excitation 360 nm/Emission 460 nm). IC₅₀ values and curve fits were obtained using (GraphPad Prism Software, Inc.).

HDAC6 inhibition assay

HDAC6 inhibitory activity was determined for each compound using the Fluorogenic HDAC6 assay kit from BPS Bioscience (Catalogue No: 50077). Assays were performed according to the manufacturer’s instructions as described below. This assay involves a two-step reaction: first, the substrate with the acetylated peptide was incubated with HDAC6 enzyme, to produce the deacetylated product, which was then digested in the second step by the addition of developer (Catalogue No: 50030) to produce the fluorescent signal proportional to the amount of deacetylated substrates. Reactions were carried out in 96-well plates (Nunc low binding, black microtiter plates) and compounds were screened at 10 concentrations using 3-fold serial dilutions to generate IC₅₀s. 50 μl reactions containing HDAC assay buffer (Catalogue No: 50031), 0.1 mg mL⁻¹ BSA, 200 μM HDAC substrate 3, and varying concentrations of inhibitor were initiated by the addition of HDAC6 (final [HDAC6] = 35 ng) and incubated at 37°C for an hour. After incubation, the reactions were quenched by the addition of 50 μL of undiluted 2x developer and incubated at room temperature for 15 min. The fluorescence was measured using a Tecan infinite M200 reader (excitation, 355 nm; emission, 460 nm). IC₅₀ values and curve fits were obtained using GraphPad Prism 5.0. (GraphPad Software, Inc.). Data are representative of at least two independent experiments.

Thermal shift assay

The melting temperature (T_m) of human LSD1 and HDAC8 proteins were determined by fluorescence based thermal shift assay. Reaction mixture containing 0.5 μM protein, 5X SYPRO™ Orange Protein Gel Stain (Thermo Fisher Scientific Cat: S6650), compound at 10-fold excess for hLSD1, 15-fold excess for hHDAC8. The final volume made up with respective protein buffers. All experiments were done in triplicates at 50 μL reaction volume. The samples heated from 15°C to 95°C with an increment of 1°C per 30sec using CFX96™ Real-Time PCR Detection system–C1000 Thermal Cycler (Bio-Rad). Filters custom configured to the optimal excitation (492 nm) and emission (610 nm) wavelengths for SYPRO orange dye. Protein denaturation monitored by the increase in fluorescence signal emitted by SYPRO Orange. The SYPRO Orange binds to the exposed hydrophobic regions of protein undergoing thermal denaturation and corresponding fluorescence emissions are recorded as melt curves. The temperature corresponding to the inflection point, Tm is determined by calculating the first derivative from melt curve using CFX manager 3.1 software (Bio-Rad).

Cell treatment with compounds

Test and reference compound stocks were prepared in 100% DMSO as single use aliquots and stored at -20 °C. These stock solutions were added to culture medium to achieve the desired test concentrations. DMSO concentration was kept uniform across the plate and did not exceed 1%.

Cell proliferation assay

The effect of JBI-097 on cell proliferation of a panel of tumor cell lines from both haematological and solid tumor origin, was assessed using Alamar Blue-based assay (Thermofischer scientific) as per the manufacturer’s instructions. Cells were seeded in NUNC 96-well tissue culture plates (Life Technologies) in 100 mL culture medium and incubated at 37°C/ 5% CO₂ to adhere to plastic for 18–24 h. After 16–24 h, compounds were added to the cells at 10 concentrations ranging from 10–0.0005 μM prepared in 3-fold serial dilutions. Cells were incubated for 68–72 h at 37°C/ 5% CO₂. One set of plates (designated for the 144 h read out) were centrifuged at low speed for 30 seconds, and respective compounds were replenished in fresh media and incubated for another 68–72 h at 37°C/ 5% CO₂. At the end of incubation period, Alamar Blue™ reagent was added and incubated for 1–3 h at 37°C/ 5% CO₂ and fluorescence was read on a Tecan M200 plate reader at 540 nm excitation, 590 nm emission wave lengths. The cellular GI₅₀ value corresponds to the concentration at which 50% growth inhibition was achieved. The inhibition curve was then fitted by plotting % inhibition against log concentration in GraphPad Prism 5.0 (GraphPad Software, Inc.) and the GI₅₀ value of compound was determined. Data are representative of at least two independent experiments.

Western blot

Cells were harvested and washed twice with cold PBS after the indicated treatments. Cell pellets were lysed in 30μl of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 12,000 ×g for 20 min at 4°C, and the protein content in the supernatants was determined using the Bradford reagent (Bio-Rad). Equal amount of protein lysate was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk powder in PBS-Tween 20 buffer for 1 h, followed by incubation with primary antibody at 4°C overnight. Membranes were washed in PBST and then incubated with HRP-conjugated anti-goat or mouse anti- IgG (Cell Signalling Technology) antibody and detected using the Millipore ECL Western Blot substrate. β-actin was used as a loading control. Antibodies were obtained from the following sources: Acetyl-α- Tubulin (Cell Signalling Technology, 3971S, 1:500), β-actin (Santa Cruz Biotechnology, sc47778, 1:2000), HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:2000 for β-actin blots, 1:1000 for all others). Blots shown are representative of at least two independent experiments.

Quantitative real time-PCR

Total RNA was isolated using the TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) reagent according to the manufacturer’s instructions. The obtained RNA was reverse-transcribed to synthesize the complementary DNA from RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.S.). DyNAmo ColorFlash SYBR Green qPCR Kit (Thermo Fisher Scientific) was used with cDNA and gene specific primers at a final volume of 10 μL. Real-time quantitative PCR was performed for 40 cycles of 15 s each, at 95°C and 30 s at 60°C, using a CFX96 real time machine (Bio-Rad). All reactions were performed in duplicate and the data was calculated using the delta (delta Ct) method. The relative gene expression levels for a target gene was normalized to the expression of β-actin. All the primer sequences were procured from Eurofins Genomics India Limited. Primer sequences for the analysed genes are as follows:

1. beta Actin F: 5′-GTGGGGTCCTGTGGTGTG-3′, beta Actin R: 5′-GAAGGGGACAGGCAGTGA-3′. Gfi1b F 5′-CCTCTTGTGCCCAGCACT-3′, Gfi1b R: 5′- CGTGAGGGGTGGAGAAGAC-3′, CD86 F: 5′- ACAGCAGAAGCAGCCAAAAT-3′, CD86 R: 5′- GAATCTTCAGAGGAGCAGCAC-3′, CD11bF: 5′-GGCATCCGCAAAGTGGTA-3′, CD11b R: 5′-GGATCTTAAAGGCATTCTTTCG-3′

In vivo studies

To evaluate the efficacy of JBI-097 in vivo, tumor xenograft models were developed in female SCID beige mice (6–8 weeks old, Vivo Biotech, India [under the license from Taconic]) by injecting subcutaneously with 5 × 10⁶ erythroleukaemia cells (HEL92.1.7) or Multiple Myeloma cells (MM1.S) in 100 μL of serum-free medium mixed with 1:1 ratio of matrigel (BD, USA). When the tumors reached an approximate volume of 100 mm³, the mice were randomized into different treatment groups with a comparable average tumor volume across the groups using the standard = RAND() function in Microsoft Excel.

Mice injected with HEL92.1.7 were either treated with dosing vehicle (0.5% methylcellulose) or JBI-097 at 25 and 50 mg/kg; PO; QD. Mice with MM1.S tumors were either treated with dosing vehicle (0.5% methylcellulose), JBI-097 12.5 mg/kg; PO; QD, Pomalidomide (2.5 mg/kg, PO for 5 days per week), Bortezomib (0.5 mg/kg; Intraperitoneal IP; twice a week, BIW), the combination of JBI-097 and Pomolidamide or combination of JBI-097 and Bortezomib. For CT-26 syngeneic mouse colon cancer model female BALB/c (6–8 weeks old, Vivo Biotech, India) were used. 1x10⁶ CT26 tumor cells in 100 μL of serum-free medium mixed with 1:1 ratio of matrigel were injected s.c. into the right flank of mice. When tumors reached 70–100 mm³; mice were randomized into various treatment groups and dosed either with vehicle control (0.5% methylcellulose) or JBI-097 (20 mg/kg; PO; for 5days on and 2 days off), or anti-mouse PDL-1 antibody (BioXcell) (i.p. at 100 μg per mice Q4D). During the experiment, body weights were measured 2–3 times a week and tumor volume was measured using callipers every other day in three dimensions. Tumor volume was calculated from the pi-based ellipsoid volume formula π/6*length*width*height. Mice were culled when the tumor volume reached either the ethical limit of 2000 mm³ or if the tumor was ulcerated. The percentage change in tumor volume from baseline was used to assess the response to treatment. Tumor measurements were performed in a blinded manner throughout the study.

Biochemical potency of JBI-097

Compounds targeting LSD1 and HDAC6 were designed using both SBDD and rationale approach with a clear understanding of specific pharmacophore requirements of the LSD1 inhibition and HDAC inhibition. Several compounds hypothesized to have LSD1 and HDAC6 inhibitory activity were synthesized and profiled in LSD1 and HDAC6 enzyme activity assay. The structure of one of the leads JBI-097 is depicted in the Fig 1A. We recently published the results of the SAR analysis, PK profile and synthetic methodology of JBI-097 and other various compounds [29]. Here in we describe the biological characterization of the lead molecule JBI-097.

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Fig 1. Structure, biochemical activity and melt curve of JBI-097.

(1A) Chemical Structure of JBI-097. (1B) JBI-097 potently inhibited LSD1 in TR-FRET Assay. The results shown are representative from two independent experiments. (1C and 1D) Derivative melt curves of 1C. LSD1 and 1D. HDAC8 in the presence of JBI-097 or DMSO. Experiments were done in triplicates.

https://doi.org/10.1371/journal.pone.0279063.g001

JBI-097 showed a strong potency in inhibiting LSD1 and HDAC6 enzymatic activities with an IC₅₀ of 6 (Fig 1B) and 48 nM, respectively. JBI-097 was screened against a panel of HDACs comprising of HDAC1 to HDAC10 (Table 1) (Reaction Biology corp., Malvern, USA) where it showed strongest activity against HDAC6 with an IC₅₀ of 48 nM, followed by HDAC8 (IC₅₀: 71 nM). Against the remaining HDACs it showed an IC₅₀ of about 1.1 μM to >10 μM with ~48 to ~70-fold selectivity against HDAC1 as compared to HDAC8 and HDAC6 IC₅₀s, respectively. Therefore, it was clear that JBI-097 was a HDAC6/8 selective inhibitor. Ricolinostat, another reported HDAC6 inhibitor that is in clinical trials also showed strongest potency against HDAC6, and its potency on class I HDACs was in the range of 50 nM.

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Table 1. HDAC Isoform Selectivity of JBI-097.

https://doi.org/10.1371/journal.pone.0279063.t001

For further confirmation of JBI-097 binding to LSD1, thermal shift assays were carried out by SYPRO-Orange dye incubation followed by PCR to assess the melting temperature (T_m). T_m of full length LSD1 protein in the absence of compound was 43°C whereas when incubated with JBI-097 it shifted to 55°C suggesting that binding of the compound stabilized the protein (Fig 1C). Similarly, when JBI-097 was incubated with full length HDAC8, clear shift in Tm (41°C to 47°C) was observed (Fig 1D).

Anti-cancer activity

JBI-097 when tested in a panel of non-small lung cancer, melanoma, and hepatocellular carcinoma tumor cell lines for 6 days, showed a strong anti-proliferative activity with EC50 values ranging from 1.06 to 8.09 μM (Table 2). Similarly, JBI-097 inhibited the proliferation of haematological cancers like acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML), erythroleukaemia, and multiple myeloma with stronger potency with EC50 values ranging from 0.002 μM to 1.5 μM (Table 2).

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Table 2. Anti-proliferative activity of JBI-097 in various cancer cell lines.

https://doi.org/10.1371/journal.pone.0279063.t002

To ascertain that both LSD1 and HDAC6 mechanisms are operating, 3 day and 6-day proliferation assays were carried out with JBI-097 along with LSD1 (Iadademstat) and HDAC6 (Ricolinostat) selective inhibitors. With the dual inhibitor JBI-097, in HEL92.1.7 erythroleukaemia cells, anti-proliferative activity was observed even at 3 days of incubation with an EC50 of 0.722 μM similar to the HDAC6 inhibitor, suggesting that the initial activity observed could be through HDAC6 inhibition. LSD1 selective inhibitor did not show any measurable anti-proliferative activity at 72 h but showed a strong anti-proliferative activity at 6 days of incubation; but interestingly, cell growth inhibition saturated at 50–60% and complete inhibition was not achieved even at a very high concentration of 10 μM. With HDAC6 inhibitor Ricolinostat, comparable anti-proliferative activity was observed both at 72 and 144 h of treatment with an EC50 of 4.4 and 3.7 μM, respectively. JBI-097 on the other hand showed a strong anti-proliferative activity with 100% cell growth inhibition on day 6 with an EC50 of 0.049 μM. This strong increase in potency at 6 days of treatment suggests that the LSD1 mechanism is operational in the dual inhibitor (Fig 2). These results clearly suggest that both LSD1 and HDAC6 mechanisms are targeted by the dual inhibitor.

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Fig 2. Anti-proliferative activity of JBI-097 in human erythroleukemia HEL 92.1.7 cells.

Cell proliferation was evaluated using a Alamar Blue assay. Human cell lines (HEL 92.1.7; density, 5 and 10 × 10³ for 3 days and 6 days; respectively) were incubated with or without the indicated concentrations of JBI-097, Ricolinostat and Iadademstat for 72 and 144 h. The results shown are representative from two independent experiments.

https://doi.org/10.1371/journal.pone.0279063.g002

Biomarker studies

We further carried out biomarker studies to understand the target engagement of the dual inhibitor on both the targets. Erythroleukemia cell line, HEL92.1.7 was treated for different durations (3–48 h) with the dual inhibitor and differentiation markers GFI1b, CD11b and CD86 were assessed. JBI-097 showed a strong and sustained up-regulation of these markers at all the time points tested. For each marker maximum modulation was observed at 24 h. Also, for some of the markers, slight decrease was observed at higher concentrations (Fig 3A–3C). Similarly, effect of JBI-097 on acetylation of α-Tubulin was assessed at various time points where a time and concentration dependent increase was observed from 3 to 48 h and representative blots of 3 h is shown in (Fig 4).

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Fig 3. JBI-097 effect on the modulation of differentiation markers in HEL 92.1.7 cells.

(3A) Effect of JBI-097 on CD11b modulation in HEL 92.1.7 Cells. Cells were treated with 8 concentrations of JBI-097, 10 and 1 μM of JBI-135 (Ricolinostat) and JBI-236 (Iadademstat) for 24h and the mRNA level of CD11b was estimated by RT-qPCR. Error bars indicate mean ± S.E, (3B) Effect of JBI-097 on CD86 modulation in HEL 92.1.7 Cells. Cells were treated with 8 point CRC of JBI-097, 10 and 1 μM of JBI-135 (Ricolinostat) and JBI-236 (Iadademstat) for 24h and the mRNA level of CD86 was estimated by RT-q PCR. Error bars indicate mean ± S.E, (3C) Effect of JBI-097 on Gfi1b modulation in HEL 92.1.7 Cells. Cells were treated with 8 point CRC of JBI-097, 10 and 1 μM of JBI-135 (Ricolinostat) and JBI-236 (Iadademstat) for 24h and the mRNA level of Gfi1b was estimated by RT-q PCR. Error bars indicate mean ± S.E.

https://doi.org/10.1371/journal.pone.0279063.g003

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Fig 4. JBI-097 inhibition increased tubulin acetylation in HEL92.1.7 cells.

HEL 92.1.7 cells were cultured with vehicle control, JBI-097 (3fold—8point dose response from 10 μM), Ricolinostat (10, 1 μM) and Iadademstat (10, 1 μM) for 3 h. Whole-cell lysates were subjected to immunoblotting with the indicated antibodies. Vinculin was used as loading control.

https://doi.org/10.1371/journal.pone.0279063.g004

Efficacy study

The anti-tumor activity of JBI-097 in vivo was examined in xenograft mouse models of erythroleukaemia (HEL92.1.7) cells or multiple myeloma cells (MM1.S). When HEL92.1.7 xenograft tumors, reached a size of ~100 ± 10 mm³, mice were randomized into various groups (n = 6) including vehicle (0.5% methyl cellulose, PO), JBI-097 25 mg/kg, and JBI-097 50 mg/kg, PO, QD. Mice were dosed for 14 days or until the endpoint tumor volume of 1200 ± 100 mm³ was achieved. As shown in Fig 5A, JBI-097 suppressed the growth of HEL92.1.7 tumors in a dose-dependent manner compared to the control treatment. The % tumor growth inhibition (TGI) of JBI-097 was 76% and 91%, for 25 and 50 mg/kg groups, respectively. Treatment at all dose levels was well tolerated with no mortality; body weight changes were within acceptable limit and a loss of around 5–10% was observed in 2 animals from 50 mg/kg group (Fig 5B).

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Fig 5. Efficacious of JBI-097 in multiple Xenograft models.

5A: SCID mice were injected s.c. with 5 × 10⁶ HEL 92.1.7 cells and were treated with vehicle, JBI-097 (25 and 50 mg/kg, po, QD), for 14 d.; n = 6 mice per group. 5B: HEL92.1.7 xenograft: Body weight monitored from Day1 to Day14; n = 6 mice per group. 5C: SCID mice were injected s.c. with 5 × 10⁶ MM1.S cells and were treated with vehicle, JBI-097 (12.5 mg/kg, po), Bortezomib (0.5 mg/kg, IP, BIW), Pomalidomide (2.5mg/kg, po, 5x/week), or Bortezomib + JBI-097 or Pomalidomide + JBI-097 for 13 d.; n = 7 mice per group. 5D: MM1.S xenograft: Body weight monitored from Day1 to Day13; n = 7 mice per group. 5E: BALB/c mice were injected s.c. with 1 × 10⁶ CT-26 cells and were treated with vehicle, JBI-097 (20 mg/kg, po), Anti PD-L1 (100μg/animal, IP, Q4D), or Anti PD-L1 + JBI-097 for 14 d.; n = 7 mice per group. 5F: CT-26 syngeneic model: Body weight data from Day1 to Day14; n = 7 mice per group. All data are represented as mean ± SE. In all the studies tumor volume was measured and calculated versus time (days).

https://doi.org/10.1371/journal.pone.0279063.g005

We further investigated the efficacy of JBI-097 in multiple myeloma MM1.s xenograft model (n = 7) in combination with Bortezomib (BTZ) and Pomalidomide. As shown in Fig 5C, while the JBI-097 as a single agent showed TGI of 23% at 12.5 mg/Kg dose, the combination of JBI-097 with BTZ led to a more potent reduction in tumor volume, with a TGI of 82%, similarly, JBI-097 showed a TGI of 60% in combination with pomalidomide. Bortezomib treatment did cause some toxicity as observed by body weight reduction and mortality of 1 mouse (1/7) Pomalidamide alone as well as in combination with JBI-097 was well tolerated throughout the study (Fig 5D).

We also further investigated the efficacy of JBI-097 as a single agent and in combination with anti-PD-L1 antibody in inhibiting the tumor growth in CT-26 mouse colon cancer derived syngeneic model (n = 7). As shown in Fig 5E, JBI-097 and anti-PD-L1 alone showed a TGI of 50 and 52%, respectively while the combination of JBI-097 with anti-PD-L1 led to a stronger TGI of 76%. All the treatment groups were well tolerated with no significant body weight loss or mortality (Fig 5F).