NEO2734

Targeting both BET and CBP/EP300 proteins with the novel dual inhibitors NEO2734 and NEO1132 

leads to anti-tumor activity in Multiple Myeloma

Katie R. Ryan§*, Francis Giles†

, Gareth J. Morgan §€

Authors affiliations:

§ UAMS Myeloma Center, Little Rock, AR, 72205

† Developmental Therapeutics Consortium, Chicago, IL 60611

Current address: € NYU Grossman School of Medicine, New York, NY 10016

* Corresponding author: KRR Address: UAMS Myeloma Center, 4301 W. Markham St, Little Rock, AR,

72205 Email: [email protected] Phone: +1-501-257-7872 Fax: +1-501-526-2873

Running Title:

Dual inhibition of BET & CBP/EP300 in MM

Novelty Statement:

Two promising epigenetic therapeutic targets have emerged for the treatment of hematologic malignancies,

BET and CBP/EP300 proteins, here we investigate the effect of the novel dual inhibitors, NEO2734 and

NEO1132, on multiple myeloma (MM). The dual inhibitors exhibited a strong anti-tumor effect across a

panel of MM cell lines when compared to single inhibitors, with sensitivity correlating with the expression

levels of c-MYC and not molecular subgroups. Our data indicates the potential value of the novel dual

inhibitors, NEO2734 and NEO1132, as a single drug double hit approach for treating a broad range of MM

and warrants further clinical investigation.

Keywords: Multiple Myeloma BET/BRD4 CBP/EP300 Dual inhibitor

Word count:

Abstract 188. Main text 4000. Number of tables 2. Number of figures 4. Number of references 48.

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ABSTRACT

OBJECTIVES

Two promising epigenetic therapeutic targets have emerged for the treatment of hematologic malignancies,

BET and CBP/EP300 proteins. Several studies have shown that targeting these individual classes of

proteins has anti-tumor activity in multiple myeloma (MM), as well as other cancers. Here we present the

first data exploring the anti-tumor activity of two novel dual inhibitors, NEO2734 and NEO1132, of both BET

and CBP/EP300 proteins in MM.

METHODS

16 MM cell lines (MMCLs) were treated with the dual inhibitors NEO2734 and NEO1132, the single BET

inhibitors JQ1, OTX015, IBET-762 and IBET-151, and a single CBP/EP300 inhibitor CPI-637.

RESULTS

The dual inhibitor NEO2734 showed strong anti-tumor activity and was consistently highly active against all

MMCLs, being as potent as JQ1 and more so than other single inhibitors. NEO2734 and NEO11132

induced a significant G1 cell cycle arrest and decreased c-MYC and IRF4 protein levels in MMCLs

compared to the other single inhibitors. Sensitivity to the dual inhibitors was not dependent on a specific

MM molecular subgroup but correlated with c-MYC protein expression levels.

CONCLUSIONS

The dual inhibition of BET and CBP/EP300 has potential therapeutic benefits for patients with MM.

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INTRODUCTION

Multiple myeloma (MM) is a malignancy of neoplastic plasma cells characterized by the production

of a paraprotein and organ damage including bone fractures, renal failure, anaemia, and hypercalcemia 1,2

It constitutes 15% of all hematologic malignancies1

. The primary tumor-initiating genetic events include

translocations and hyperdiploidy 3,4. Progression of MM corresponds with the accumulation of secondary

incurable malignancy, with nearly all patients succumbing to treatment-refractory disease 2,10

Epigenetic mechanisms regulate gene expression without alterations in DNA sequence. Disruption

of these epigenetic processes leads to altered gene function and contributes to the pathogenesis of MM

11,12. Bromodomain and extra-terminal domain (BET) proteins comprise a family of four related proteins

(BRD2, BRD3, BRD4, and BRDT) that are chromatin adaptors binding covalently to acetylated lysine

residues of histones and influencing gene transcription 11. BET inhibitors are emerging as promising target

molecules for the treatment of both solid and hematologic malignancies 13–15. JQ1 and IBET-762 were two

of the first BET inhibitors to be described and have been shown to suppress proliferation of MM cells

13,14,16,17. Although most BET inhibitors are pan inhibitors their effects are mostly attributed to the inhibition

of BRD4. BRD4 has a direct role in the regulation of the transcription of genes involved in cell cycle

regulation and oncogenes, such as c-MYC. BRD4 binds to acetylated histones during the M/G1 transition

in the cell cycle promoting progression to S phase 2,18. Depleting BRD4 levels by siRNA inhibits cell cycle

progression, due to G1 cell cycle arrest 18,19. BET inhibitors prevent BRD4 from binding to chromatin by

competitively binding to the acetyl-lysine recognition pocket of BET bromodomains 13,17. This displacement

of BRD4 from chromatin leads to the suppression of c-MYC through the inhibition of both MYC transcription

and MYC-dependent oncogenic programs 13–15,17,20. Increasing numbers of BET inhibitors are being

reported and a number of groups have investigated their effect on MM 11,16,21,22

The cyclic AMP response element binding protein (CREB)-binding protein (CBP) and E1A

interacting protein of 300 kDa (EP300) are closely related bromodomain-containing histone

acetyltransferases (HATs) that act as transcriptional coactivators, which regulate a number of important

cellular events 23,24. Due to the involvement of CBP and EP300 in many signaling pathways a number of

studies have implicated CBP/EP300 in cancer 25–30. Focus has now been placed on CBP/EP300

bromodomain inhibition as it may represent a new therapeutic strategy to treat human malignancies 25,29,30

Conery et al, were the first to show that MMCLs were strongly affected by CBP/EP300 inhibitors observing

approach to restore immunomodulatory drug sensitivity in MM 32

Oncogenic MYC activation and overexpression is a common secondary event in MM 33,34. Both

BET and CBP/EP300 single inhibitors display promising results as MM agents based on downregulation of

c-MYC protein levels via different pathways resulting in G1 cell cycle arrest 13–15,17,20,31. Based on this data

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we investigated whether a dual BET and CBP/EP300 inhibitor would be more effective. Here we present

the first data exploring the anti-tumor activity of the novel dual inhibitors of both BET and CBP/EP300,

NEO2734 and NEO1132, in MM. We observed a strong anti-tumor effect across a panel of 16 MMCLs

following treatment with either NEO2734 or NEO1132 with NEO2734 being is as potent as JQ1 and more

so than other single BET and CBP/EP300 inhibitors. The dual inhibitors induced more significant G1 cell

cycle arrest following treatment by comparison with single BET or CBP/EP300 inhibitors, and more

effectively decreased both c-MYC and IRF4 protein expression. We observed no association between

molecular subgroups and sensitivity to the dual inhibitors suggesting they maybe broadly active. We show

that sensitivity to the dual inhibitors may correlate with the expression levels of c-MYC. Taken together,

these data indicate that dual inhibition of BET and CBP/EP300 has potential therapeutic benefits for

patients with MM.

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MATERIAL AND METHODS

Cells lines and inhibitors

16 human multiple myeloma cell lines (MMCLs) were used in this study: U266, RPMI8226, OPM2,

MM1S, LP1, L363, KMS28PE, KMS28BM, KMS18, KMS12PE, KMS12BM, KMS11, KMM1, JJN3, H929

and EJM. Cell lines were grown in RPMI 1640 medium (ThermoScientific) containing 10% heat-inactivated

foetal calf serum (Hyclone Laboratories) in a humidified incubator (37oC, 5% CO2). JQ1 (HY-13030),

OTX015 (HY-15743), IBET-151 (HY-13235), IBET-762 (HY-13032) and CPI-637 (HY-100482)

(MedChemExpress). NEO2734 and NEO1132 were provided by Epigene Therapeutics Inc. Quebec

Canada. All compounds were reconstituted in DMSO.

Cell counting/viability assay and analysis

MMCLs were seeded at 5×103

cells/well and incubated with either DMSO or inhibitor (full

concentration range used 0.005-50 µM, depending on inhibitor and MMCL) for 72 hours at 37oC. Cell

viability was analyzed by performing a CCK8 assays following manufactures instructions (Dojindo

Inc.). Cells were plated in duplicate/per condition and at least n=3 assays were performed. IC50 was

generated using nonlinear regression (curve fit) with Log(inhibitor) vs. normalized response with variable

slope and interpolation of unknown values on X= log(X) transformed data. Mean and median IC50 was

analyzed and statistical significance was generated using Mann-Whitney tests. The mean IC50s for each

MMCL treated with NEO2734 for each molecular subgroup were grouped and compared against the mean

of the others IC50s using a Mann-Whitney test.

Cell cycle analysis

MMCLs were seeded at 2×105

cells/well and incubated with DMSO or inhibitor for 24 hours

(concentration indicated in Figures). Cells were washed with phosphate buffered saline (PBS), fixed and

permeabilized with 70% ethanol for 1 hour at -20oC, washed with PBS, treated with RNAse A (final

concentration 0.5 mg/ml) for 1 hour at 37oC, stained with propidium iodide (final concentration 10 µg/ml),

incubate at 37oC for 1 hour. Cells were subjected to flow cytometry using the FACSVerse (Becton

Dickinson). Data were analyzed with FC Express software (De Novo). The mean percentage, +- standard

error of the mean (SEM), of cells in each phase of the cell cycle was analyzed, from n=4 assays. Mann

Whitney test were performed for G1 comparison.

Western blotting, antibodies and densitometric analysis

Myeloma cells were seeded 1×106

cells/well and incubated with either DMSO or compound

(concentration indicated in Figures) for 16 hours. Cells were collected by centrifugation, washed in ice cold

PBS twice, lysed in RIPA buffer (ThermoScientific) containing HALT phosphatase inhibitors

(ThermoScientific) and sonicated. 15 µg of protein was loaded onto a 4-12% gel and transferred to an

IBlot2 nitrocellulose membrane (Invitrogen). Primary antibodies were anti-c-MYC (13987 Cell Signaling),

anti-IRF4 (15106 Cell Signaling) anti--actin (A5441 Sigma). Secondary antibodies were horseradish

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peroxidase-conjugated anti-rabbit and anti-mouse (Cell Signaling). Protein expression was detected using

the ECL substrate (ThermoScientific). Densitometry: Protein band intensities were determined using

ImageJ and the data presented as the mean adjusted volume intensity ratio of protein of interest to -actin

(loading control), +- SEM, correlation analysis was performed from at least n=3 experiments using Pearson

correlation coefficients with a two-tailed P-value.

Statistical Analysis

All statistical analysis described above was carried out using PRISM (GraphPad) and statistical

significance was defined as P less than 0.05; P value = * < 0.05, ** <0.01, *** <0.001, **** <0.0001, n.s not

significant.

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RESULTS

The dual inhibitors NEO2734 and NEO1132 are potent anti-myeloma drugs and have strong anti￾tumor activity across a panel of MMCLs

MM cell lines (MMCLs) were treated with increasing concentrations of the dual inhibitors NEO2734

and NEO1132 as well as the single BET inhibitors JQ1, OTX015, IBET-762 and IBET-151, and to a

CBP/EP300 inhibitor CPI-637 for 72 hours. The mean IC50 values were calculated for the compounds in

each of the MMCLs. When comparing the IC50 across the different MMCLs JQ1 ranked as consistently the

most potent followed by NEO2734 for each of the MMCLs tested (Table 1. Figure 1A-B). For NEO2734

the mean IC50 range for 15/16 MMCLs (excluding U266 cells) was 0.05-0.32 µM giving a 6.2 fold

difference in concentration, the main outliers were U266 cells which have a mean IC50 of 1.24 µM and a

24.8 fold difference in concentration from the most sensitive MMCL (Table 1). U266 was the most resistant

MMCL for each of the compounds tested. If we exclude U266 cells and calculate the fold change of the

mean IC50 concentration for each of the inhibitors they reach IC50 for 15/16 MMCLs within a relatively

small concentration range except CPI-637: NEO2734 6.2, NEO1132 6.3, JQ1 12.7, OXT015 17.7, 1BET-

151 8.0, IBET-762 15.3 and CPI-637 93.4. Together these data suggest that the dual inhibitors, NEO2734

and NEO1132, are more effective at decreasing cell viability of different MMCLs in a smaller concentration

range.

Both NEO2734 and NEO1132 showed anti-tumor activity across all 16 MMCLs and decreased cell

viability with a median IC50 of 124 nM (95% C.I., 52-264 nM) and 375 nM (95% C.I., 161-751 nM)

respectively, with NEO2734 being significantly more potent that NEO1132 (P = 0.0039). As a comparison,

all MMCLs were exposed to the single BET inhibitors JQ1, OTX015, IBET-762 and IBET-151, and to a

CBP/EP300 inhibitor CPI-637, the median IC50 values of these compounds were 70 nM (95% C.I., 33-144

nM), 243 nM (95% C.I., 114-457 nM), 340 nM (95% C.I., 176-930 nM), 668 nM (95% C.I., 283-1104 nM)

and 1912 mM (95% C.I., 508-4818 nM) respectively. The novel dual inhibitor NEO2734 was more potent

than the single BET inhibitors OTX015 (P = 0.0424), IBET-762 (P = 0.0034), IBET-151 (P = <0.0001) and

the CBP/EP300 inhibitor CPI-637 (P = <0.0001), and showed no significant difference from the highly

potent JQ1 inhibitor (P = 0.079) (Figure 1C). These data suggest that NEO2374 and NEO1132 have

strong anti-tumor activity and are highly effective on a range of MMCLs.

Dual inhibitors NEO2734 and NEO1132 decrease c-MYC and IRF4 protein expression

BET inhibitors prevent BRD4 from binding to chromatin, which leads to the suppression of c-MYC

by the inhibition of both MYC transcription and MYC-dependent oncogenic programs 13–15,17,20. CBP/EP300

inhibitors prevent histone acetylation and have been shown to directly suppress expression of the

transcription factor IRF4 and the subsequent down regulation of c-MYC 31. We observed a decrease in the

levels of proteins regulated by BET and CBP/EP300, c-MYC and IRF4, in MMCLs following 16 hour

exposure to the dual inhibitors indicating these inhibitors are affecting their respective target regulatory

domains (Figure 2). In H929, KMS12PE and JJN3 cells treated with the dual inhibitors NEO2734 and

NEO1132 there is a marked decrease in c-MYC protein levels when compared to the DMSO control and

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the single BET and CBP/EP300 inhibitors, but comparable to c-MYC levels of cells treated with JQ1

(Figure 2). In KMS12PE cells treated with the CBP/EP300 inhibitor, CPI-637, there is a marked decrease

in IRF4 protein levels compared to control cells, no decrease in IRF4 protein levels were observed in the

KMS12PE cells treated with the dual inhibitors or the single BET inhibitors and an increase in IRF4

expression was observed in KMS12PE cells treated with the BET inhibitor JQ1 (Figure 2D, F). In both

H929 and JJN3 cells IRF4 protein levels decreased when compared to the DMSO control and the single

BET inhibitors but were comparable to IRF4 levels in CBP/EP300 inhibitor treated cells (Figure 2A, C, G,

I). These data indicate that the dual inhibitors, NEO1132 and NEO2734, deplete c-MYC and IRF4 protein

levels suggesting they are inhibiting their intended targets BRD4 and CBP/EP300.

Dual inhibitors NEO2734 and NEO1132 induce a G1 cell cycle arrest

BET inhibitors induce G1 cell cycle arrest by preventing BRD4 from binding to acetylated histones

of genes involved in the M/G1 transition in the cell cycle which prevents progression to S phase 2,18

CBP/EP300 inhibitors induce a G1 cell cycle arrest and in MM cells this has been attributed to the direct

suppression of the transcription factor IRF4 and the subsequent down regulation of c-MYC 31. Like the

single BET and CBP/EP300 inhibitors, NEO2734 and NEO1132 induce a G1 cell cycle arrest following 24

hours of treatment (Figure 3). All inhibitors induced a significant (P < 0.05) G1 arrest in H929, KMS12PE

and JJN3 cells at 24 hours when compare to DMSO treated control cells. An exception to this feature is

KMS12PE cells treated with the CBP/EP300 inhibitor, CPI-637, and JJN3 cells treated with IBET-762, as

the percentage of cells in G1 was not significantly different (P = 0.83 and 0.15) from DMSO treated control

cells. The dual inhibitor NEO1132 only induced a significantly larger (P = 0.008) percentage of JJN3 cells to

be in G1 arrest when compared to the single BET inhibitor IBET-762. However, the dual inhibitor NEO2734

induced a significantly larger percentage of JJN3 cells to be in G1 arrest when compared to IBET-151,

IBET-762 and CPI-637 (P = 0.03, 0.008 and 0.03) but was not significantly different from JJN3 cells

treated with OXT015 or JQ1 (P = 0.15 and 0.8) (Figure 3C). The dual inhibitor NEO1132 induce a

significantly larger (P < 0.05) percentage of H929 and KMS12PE cells to be in G1 arrest when compared to

each of the single inhibitors except JQ1 (P = 0.057), however, the dual inhibitor NEO2734 induce a

significantly larger (P < 0.05) percentage of cells to be in G1 arrest when compared to each of the single

inhibitors (Figure 3A-B). These data indicate that the dual inhibitors NEO2734 and NEO1132 induce a

strong G1 cell cycle arrest in MMCLs and that NEO2734 is more effective, in some cases, in inducing G1

arrest than the highly potent single BET inhibitor JQ1.

Dual inhibitors show heterogeneity in sensitivity across MMCLs

Sensitivity to compounds differs for each MMCL with some being more sensitive than others

(Table 1). The general trend of most sensitive to most resistant MMCLs for the dual inhibitors NEO2734

and NEO1132 is very similar to that of the single BET inhibitors, however this trend does not hold when

comparing these groups to the CBP/EP300 inhibitor, CPI-637 (Table 1). KMS12PE and KMS28PE cells

are very sensitive to the dual and single BET inhibitors (mean IC50 0.03-0.31 µM and 0.04-0.28 µM

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representatively) but are two of the more resistant cell lines when treated with CPI-637 (mean IC50 3.06

and 4.82 µM). Similarly, RPMI8226 and L363 cell lines are more resistant to the dual and single BET

inhibitors (mean IC50 0.14-1.1 µM and 0.17-1.03 µM) but are two of the more sensitive cell lines when

treated with CPI-637 (mean IC50 0.76 and 0.36 µM). Although RPMI8226 and L363 are more resistant in

the group rankings of the dual inhibitors compared to the group rankings of CPI-637, the dual inhibitors are

still more effective at decreasing viability than that of the CBP/EP300 inhibitor alone.

The primary tumor-initiating genetic events of MM involve one of five primary translocations which

leads to the altered expression of specific oncogenes 3,4. Translocations have different prevalence rates,

treatment regimens and prognostic outcomes 3

. Each of the primary translocation groups is represented in

our panel of 16 MMCLs (Table 2). To ascertain if a specific translocation group is more susceptible to the

dual inhibitors the mean IC50s for each MMCL treated with NEO2734 with a particular translocation group

were grouped and compared against the mean IC50s of MMCLs not harboring that specific translocation

group for example, t(4:14) group vs non- t(4:14) group. No statistical difference was associated with

NEO2734 sensitivity and the common primary translocations in the MMCLs. There was also no statistical

difference associated with NEO2734 sensitivity and groups associated with good prognostic outcome

(t(11;14), t(14;16) and t(6;14)) compared to groups associated with poor prognostic outcome (t(4;14) and

t(14;20)) 3

. These data indicate that the dual inhibitors are not limited to treating a specific molecular

subgroup of MM based on translocation group.

Sensitivity to dual inhibitors correlates with c-MYC but not IRF4 protein levels

Progression of MM corresponds with the accumulation of secondary genetic and epigenetic

alterations such as abnormal activity of several transcription factors including c-MYC and IRF4 5,8,9. Both

BET and CBP/EP300 inhibitors affect cell viability by depleting either directly or indirectly c-MYC protein

levels 13–15,17,20,31. To ascertain if elevated protein levels of either c-MYC or IRF4 correlates with sensitivity

to the dual inhibitors we compared c-MYC and IRF4 protein expression levels in our panel of 16 MMCLs.

Across the 16 MMCLs there is variation in both c-MYC and IRF4 protein expression but most MMCLs in the

panel express both c-MYC and IRF4 (Figure 4). However, U266 cells have a significantly low level of c￾MYC compared to the other MMCLs, for example U266 cells express significantly less c-MYC (P= 0.0022)

compared to the second lowest c-MYC expressing cell line MM1S, this is consistent with previous reports

35,36. A significant correlation between sensitivity of MMCLs to dual inhibitors and c-MYC expression was

observed: NEO2734 P= 0.021 r= -0.57 and NEO1132 0.020 -0.57 (Figure 4D). This correlation was also

observed for the single BET and CBP/EP300 inhibitors as well: JQ1 P= 0.016 r= -0.59, OXT015 0.024 -

0.56, IBET-762 0.03 -0.54, IBET-151 0.019 -0.58, and CPI-637 0.047 -0.50. No significant correlation was

observed between sensitivity of MMCLs to dual inhibitors or the single inhibitors and IRF4 expression

(Figure 4D). These data suggest that these inhibitors are most effective in cell lines expressing c-MYC.

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DISCUSSION

Epigenomic abnormalities have been shown to contribute to the pathogenesis of MM, and two

promising epigenetic targets have emerged for the treatment of hematologic malignancies, BET and

CBP/EP300 proteins, with single BET and CBP/EP300 inhibitors showing promising anti-tumor activity

against MM 11–14,16,17,21,22,31,32. In this study we show that the novel dual inhibitors of both BET and

CBP/EP300 37, NEO2734 and NEO1132, show strong anti-tumor activity across a range of MMCLs, with

NEO2734 being as potent as JQ1 and more so than other single BET and CBP/EP300 inhibitors. The dual

inhibitors induced a significant G1 cell cycle arrest following treatment when compared to other single BET

and CBP/EP300 inhibitors. Spriano et al has shown that the dual inhibitors target both BET and

CBP/EP300 and we show a decrease in both c-MYC and IRF4 protein expression, indicating that

NEO2734 and NEO1132 are inhibiting their intended targets 37. By inhibiting multiple targets, the

expectation would be the dual inhibitors would be significantly more potent due to additive toxicity.

NEO2734 shows a similar IC50 to JQ1, a single BET inhibitor, but is significantly more potent than all the

other single inhibitors tested here. The dual inhibitors showed significantly more G1 arrest than the other

single inhibitors including JQ1. These data would suggest that the dual inhibition is greater than single

inhibition. JQ1 is the prototype BET inhibitor, which is why we included it in our study, however the toxicity

profile of JQ1 limits its clinical use in humans and JQ1 has been shown to be poorly tolerated in mice with

significant body weight loss leading to early euthanasia 13,38,39. Due to these issues several JQ1 derivatives

have been developed for clinical applications, among them are OTX015 and I-BET762, which we also

included in our study, these have been shown to be tolerated well in early phase clinical trials as therapies

for hematologic malignancies including MM (see ClinicalTrials.gov). Unlike JQ1, NEO2734 is tolerated well

in mice indicating, although highly potent, NEO2734 has the potential to be taken forward into clinical trials

37,40,41

The primary tumor-initiating genetic events of MM involve either hyperdiploidy or one of five

primary translocations which leads to the altered expression of specific oncogenes, these are as follows:

t(4;14) (MMSET and FGFR3), t(6;14) (CCND3), t(11;14) (CCND1), t(14;16) (MAF) or t(14;20) (MAFB) 3,4

.

Hyperdiploidy and translocations have different prevalence rates, treatment regimens and prognostic

outcomes for example: hyperdiploidy is generally considered with a favorable outcome, t(11;14), t(14;16)

and t(6;14) occur with a prevalence of 15%, 5% and 2% respectively and are generally associated with a

good prognosis compared to t(4;14) and t(14;20), with a prevalence of 15% and 1% respectively, that are

associated with poor prognosis 3

. In our study, we used MMCLs to analyse the effects of the dual inhibitors

as primary patient MM cells tend to enter G1 cell cycle arrest in vitro. It should be noted that most MMCLs

are from extramedullary disease or plasma cell leukemia such as JJN3 cells. Although they are not primary

patients cells, MMCLs are a commonly used tool for preclinical evaluation of MM response to compounds,

we also utilised 16 MMCLs to get a broad range of MM subgroups with each of the primary translocation

groups being represented. We observed no association between different primary translocation groups and

sensitivity to the dual inhibitors suggesting that the dual inhibitors are not limited to treating a specific

molecular subgroup of MM based on translocation group.

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Progression of MM corresponds with the accumulation of secondary genetic and epigenetic

alterations, with oncogenic MYC activation and overexpression being a common secondary event occurring

in 60% of MM patients in both hyperdiploidy and non-hyperdiploidy subgroups 3,5,9,33,34,42. MYC

translocations are associated with a poor outcome, especially when they involve an Ig partner, and the

activity of c-MYC reportedly increases with MM disease stage 3,33,43. Both BET and CBP/EP300 inhibitors

decrease cell viability by depleting c-MYC protein levels to induce a G1 cell cycle arrest either directly, by

the displacement of BRD4 from chromatin, or indirectly, via the direct suppression of transcription factor

IRF4 13–15,17,20,31. Our data suggests that sensitivity to the dual inhibitors correlates with c-MYC but not IRF4

protein levels. The only cell line to show resistance to any of the single or dual inhibitors was U266, which

express very low levels of c-MYC and instead express the MYCL gene 35,36. Despite the lack of c-MYC

expression, Suzuki et al reported that U266 cells were suspectable to JQ1 and IBET-151 treatment and

that IBET-151 decrease the expression of MYCL and induced a c-MYC independent cell cycle arrest 44

Even though U266 cells were the most resistant MMCL for all inhibitors tested here the dual inhibitors

decreased cell viability more efficiently than the single BET and CBP/EP300 inhibitors, except JQ1. These

data indicate that the dual inhibitors are effective on all of the MMCLs tested in this study and they may

have a prevalence for cells expressing elevated c-MYC.

The introduction of novel immunomodulatory drugs such as thalidomide and lenalidomide has improved the

response rate and progression free survival of MM patients 45. However, despite recent therapeutic

advances and improved survival, MM remains an incurable malignancy, with nearly all patients succumbing

to treatment-refractory disease, which is often highly proliferative 2,10. A common secondary event leading

to the progression of MM involves the increased expression of c-MYC 3,5,9,33,34,42. Inhibition of either BRD4

or CBP/EP300 has shown promising anti-tumor activity in MM, as they inhibit cell cycle progression by

decreasing c-MYC expression through distinct mechanisms 13–15,17,20,31. An increasing number of BET and

CBP/EP300 inhibitors are involved in clinical trials 46. There is also a body of data suggesting that

combinational therapy of immunomodulatory drugs and BET or CBP/EP300 inhibitors are beneficial for the

treatment of MM and can restore sensitivity to immunomodulatory drug and proteasome inhibitor resistant

MMCLs 21,32,47,48. The novel dual inhibitors, NEO2734 and NEO1132, showed strong anti-tumor activity

against all MMCLs tested. The dual inhibitors suppress both BET and CBP/EP300 which independently

regulate c-MYC expression 13–15,17,20,31,37

. Yan el al show that NEO2734 inhibits the growth of both prostate

cancer cells and SPOP-mutated prostate tumors, which exhibit high-level resistance to BET inhibitors 40

These data indicate that NEO2734 and NEO1132 would be good candidates for combinational drug studies

in MM and maybe potentially beneficial in reducing resistance in MM and restoring sensitivity to

immunomodulatory drugs in relapse/refractory MM patients. NEO2734, unlike JQ1, has been shown to be

tolerated well in pre-clinical animal models 37–41. Taken together, these data indicate the potential value of

the novel dual inhibitors, NEO2734 and NEO1132, as a single drug double hit approach for treating a broad

range of MM and warrants further clinical investigation.

AUTHOR CONTRIBUTION

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KRR conceived project, preformed experiments and analysis and wrote manuscript

FG provided compounds and edited/approved manuscript

GM conceived project and wrote manuscript

DISCLOSURE

F. Giles is a consultant to Epigene Therapeutics Inc.

ACKNOWLEDGMENTS

This work was supported by the Myeloma Center, University of Arkansas for Medical Science. The authors

are grateful to Epigene Therapeutics Inc. for providing NEO2734 and NEO1132.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon

reasonable request.

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FIGURE LEGENDS

Figure 1. Comparison of the mean IC50 for each individual MMCL treated either the single or dual

BET and CBP/EP300 inhibitors, and the anti-tumor effect of single and dual compounds on the 16

MMCLs as a group.

A-B Mean IC50 +- SEM following 72 hours exposure to increasing concentrations of inhibitors. Mean IC50

comparisons between NEO2734, JQ1 and OTX0151. C. IC50 comparisons between NEO2734 and the

single inhibitors. Each point represents mean IC50 for each MMCL, line represents median IC50 with

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interquartile range. Mann Whitney test performed; P value = * < 0.05, ** <0.01, **** <0.0001, n.s not

significant.

Figure 2. NEO2374 and NEO1134 decrease c-MYC and IRF4 expression.

A-C. H929 D-F. KMS12PE G-I. JJN3 MMCLs were treated with 0.1 µM of dual and BET single inhibitors

and 0.5 µM of the CBP/EP300 inhibitor, CPI-637, for 16 hours, lysates were then run on SDS-PAGE and

then blotted for c-MYC and IRF4 protein levels. Actin serves as loading control. DMSO serves as no

treatment control. Representative blot of n=3 experiments. Densitometric analysis of B, E, H. c-MYC and C,

F, I. IRF4 protein expression, data presented as the mean adjusted volume (Adj. Vol.) intensity ratio of

protein of interest to Actin (loading control) +- SEM.

Figure 3. NEO2374 and NEO1132 induce a G1 cell cycle arrest.

The mean percentage of cells +- SEM for each phase of cell cycle in A. H929 B. KMS12PE and C. JJN3

MMCLs treated with 0.1 µM of dual and BET single inhibitors and 0.5 µM of the CBP/EP300 inhibitor, CPI-

637, for 24 hours. Mann Whitney test performed for G1 comparison; P value = * < 0.05, ** < 0.01, n.s not

significant.

Figure 4: c-MYC and IRF4 protein expression levels in 16 MMCLs

A. Representative blots of c-MYC and IRF4 protein expression across 16 MMCLs, Actin used as loading

control. B-C. Densitometric analysis of c-MYC and IRF4 protein expression, data presented as the mean

adjusted volume (Adj. Vol.) intensity ratio of protein of interest to Actin (loading control) +- SEM. MMCLs

are ordered based on their sensitivity to NEO2734 from most resistant (Left) to most sensitive (Right). D.

Correlation analysis of c-MYC or IRF4 protein expression against each MMCL’s mean IC50 for each

compound was performed using Pearson correlation coefficients with a two-tailed P value. P value = * <

0.05. Representative blot and densiometric analysis of n= 3 experiments.

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TABLE LEGENDS

Table 1. Mean IC50 value in µM for each MMCL treated with dual as well as, single BET and

CBP/EP300 inhibitors. Multiple myeloma cell lines (MMCLs) were exposed to increasing concentrations of

inhibitors for 72 hours before being subjected to a CCK8 viability assay. MMCLs are ordered based on their

sensitivity to NEO2734 from most resistant (Top) to most sensitive (Bottom).

Table 2. Primary translocation groups of the 16 MMCL panel.

* KMS11 has two translocations t(4;14) and t(14;16). ** RPMI8226 has t(16;22) which results in MAF

overexpression. *** L363 has t(20;22) resulting in MAFB overexpression. n= number of represented

multiple myeloma cell lines (MMCLs) for each molecular subgroup.