Efficacy of the Novel Tubulin Polymerization Inhibitor PTC-028 for Myelodysplastic Syndrome

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Figure 1 The process of clone evolution from normal stem cell to AML via MDS. Normal

hematopoiesis is disrupted in MDS state before leukemogenesis.

45

Vincristine

Paclitaxel

Figure 2 Chemical structure of clinically used microtubule inhibitors (Vincristine &

Paclitaxel)

46

Figure 3 Chemical structure of PTC-028 and PTC596.

47

Figure 4 Distribution of tubulin in polymerized (P) vs. soluble (S) fractions analyzed by

immunoblotting in PTC-028-treated MDS-L cells. MDS-L cells were treated with 3 and

5 μM of PTC-028 and 1 μM paclitaxel for 4 hours. The fractions containing soluble and

polymerized tubulin were collected and separated by SDS-PAGE. The α-tubulin antibody

was used to detect tubulin by Western blotting. Band intensity was calculated using Image

lab (Bio-Rad) and was shown as means ± S.D. (n=3). *P<0.05, ***P<0.001 by the

Student’s t-test.

48

Figure 5 Growth of MDS-L and SKM-1 MDS cells and HL-60 and THP-1 AML cells

treated with the indicated concentrations of PTC-028. The numbers of viable cells on

days 3 and 6. Data are shown as means ± S.D. (n=3). *P<0.05, **P<0.01, ***P<0.001 by

the Student’s t-test.

49

Figure 6 CC50 of MDS and AML cell lines. Cells lines were treated with the indicated

concentrations of PTC-028 for 3 days in triplicate (left panels). CC50 was defined as the

concentration required to reduce cell viability by 50% and is presented in the right panel.

Cell viability was assessed by MTS assays.

50

Figure 7 Growth of primary MDS cells treated with PTC-028. CD34+ MDS cells were

cultured in the presence of SCF, TPO, IL-3, GM-CSF, and FLT3 ligand in the presence

of the indicated doses of PTC-028. Cell growth was examined by MTS assays after 48

hours in culture (left panel) or viable cells were counted on days 3 and 6 (right panel).

Data are shown as means ± S.D. (n=3). *P<0.05, **P<0.01, ***P<0.001 by the Student’s

t-test.

51

Figure 8 Caspase-Glo 3/7 values 3 days after the treatment with PTC-028. MDS cells

were treated with PTC-028 at the indicated doses in triplicate. An equal volume of

Caspase-Glo 3/7 agent was added to samples before recording luminescence. *P<0.05,

**P<0.01, ***P<0.001 by the Student’s t-test.

52

Figure 9 MTS assays showing the viability of MDS-L and SKM-1 cells treated with the

indicated doses of PTC-028 and decitabine (DAC) or azacitidine (AZA) relative to the

untreated control. Data are shown as means ± S.D. (n=3). Fa-CI plots are shown in the

lower panel of each graph. CI, combination index. Fa (fraction affected) indicates the

fraction of cells affected by the drug.

53

Figure 10 Apoptosis induced by PTC028 and/or DNA hypomethylating agents. MDS-L

cells were treated with PTC-028 and/or DNA hypomethylating agents (DAC or AZA) for

72 hours, stained with Annexin V and PI, and then analyzed by flow cytometry. Results

are shown as means ± S.D. (n=3). *P<0.05, **P<0.01, ***P<0.001 by the Student’s ttest.

54

Figure 11 Growth inhibition of MDS-L cells by PTC596. Growth curve of MDS-L cells

treated with the indicated concentrations of PTC596 (left panel) and CC50 of PTC596 in

MDS-L cells (right panel). Cells were treated with the indicated concentrations of

PTC596 for 3 days in triplicate to evaluate CC50.

55

Figure 12 MTS assays showing the viability of MDS-L treated with the indicated doses

of PTC596 and DAC relative to the untreated control (left panel) and a Fa-CI plot (right

panel)

56

Figure 13 CC50 of microtubule-destabilizing agents in MDS and AML cells. Cell lines

were treated with the indicated agents for 3 days in triplicate. Data are shown as means ±

S.D. (n=3).

57

Figure 14 Cell cycle arrest induced by PTC-028. MDS-L and SKM-1 were exposed to

PTC-028 for 72 hours at 40 and 80 nM, respectively. BrdU was added to the culture 4

hours before the analysis. Representative contour plots of BrdU incorporation (y axis)

versus DNA content assessed by 7-AAD staining (x axis) are shown in the left panels.

The proportion of cells at the indicated phase of the cell cycle is shown as means ± S.D.

(n=3) in the right panels. **P<0.01, ***P<0.001 by the Student’s t-test.

58

PTC-028

Gene Expression up

MDS-L

139

24

SKM-1

380

PTC-028

Gene Expression Down

MDS-L

33

SKM-1

76

Figure 15 Venn diagram showing the overlap of up-regulated (≥1.5-fold) or down- (≤

0.66-fold) genes between MDS-L and SKM-1 cells treated with PTC-028 relative to the

DMSO control.

59

Figure 16 Summary of the gene set enrichment in MDS-L and SKM-1 cells treated with

PTC-028 relative to non-treated cells in GSEA using RNA-seq data. MDS-L and SKM-1

cells were cultured in the presence of PTC-028 (MDS-L 30nM; SKM-1 40nM) for 72

hours. Representative GSEA plots are shown in the right panelspanels. Normalized

enrichment scores (NES), nominal p values (NOM), and false discovery rates (FDR) are

indicated.

60

Figure 17 Schematic representation of the xenograft MDS model using NOG IL-3/GMTG mice. NOG mice irradiated at a dose of 1.8 Gy were infused with 1×10 7 MDSL/Akaluc cells via the tail vein. On day 27 post-transplantation, recipient mice (n=5 in

each group) received vehicle and 12.5 mg/kg PTC-028 orally twice a week for 7 weeks.

61

Figure 18 The engraftment of MDS-L/Akaluc cells was confirmed by bioluminescence

imaging. Images of Akaluc signals in representative mice (3 mice each) are shown at

different time points during the treatment.

62

Figure 19 Quantification of photon counts from MDS-L/Akaluc cells in xenograft MDS

mice. Akaluc signals taken by a photon-counting analyzer. Data are shown as means ±

S.D. *P<0.05, **P<0.01, ***P<0.001 by the Student’s t-test.

63

Figure 20 Kaplan–Meier survival of mice. Survival was evaluated from the first day of

the treatment to death. The significance of differences between the PTC-028-treated and

vehicle-treated groups was assessed using a Log-rank test. *P<0.05, **P<0.01,

***P<0.001.

64

Figure 21 Schematic representation of the xenograft MDS model using NOG IL-3/GMTG mice. NOG mice irradiated at a dose of 1.8 Gy were infused with 1×107 MDSL/Akaluc cells via the tail vein. On day 33 post-transplantation, recipient mice (n=5 in

each group) received vehicle and 6.25 mg/kg PTC-028 orally twice a week. DAC was

administered at a dose of 0.3 mg/kg intraperitoneally 3 times per week.

65

Figure 22 The engraftment of MDS-L/Akaluc cells was confirmed by bioluminescence

imaging. Images of Akaluc signals in representative mice (4 mice each) are shown at

different time points during the treatment. Quantification of photon counts from MDSL/Akaluc cells in xenograft MDS mice. Akaluc signals taken by a photon-counting

analyzer. Data are shown as means ± S.D. **P<0.01, ***P<0.001 by the Student’s t-test.

66

Figure 23 Kaplan–Meier survival of mice. Survival was evaluated from the first day of

the treatment to death. **P<0.01, ***P<0.001 by the Log-rank test.

67

Figure 24 Body weight (BW) and hemoglobin (Hb) levels of mice. Data are shown as

means ± S.D. n.s., not significant by the Student’s t-test.

68

DMSO 48h

72h

96h

BMI-1

uH2A

H2A

B-actin

Figure 25 Level of BMI-1 and uH2A in MDS-L cells cultured in the presence of DMSO

and 40nM PTC-028 for 48h, 72h and 96h.

69

Figure 26. Apoptosis induced by PTC-028.The indicated cells were treated with PTC028 for 72 hours, stained with Annexin V and PI, and then analyzed by flow cytometry.

Results are shown as means ± S.D. (n=3). **P<0.01, ***P<0.001.

70

Table 1 Human MDS bone marrow samples.

71

Table 2 Upregulation of Signaling Pathway Induced by PTC-028 in MDS-L Cells

NAME

ES

NES

NOM p-val

FDR q-val

HALLMARK_TNFA_SIGNALING_VIA_NFKB

0.6108075

1.879353

HALLMARK_COAGULATION

0.5875037

1.7655075

0.002344156

HALLMARK_ALLOGRAFT_REJECTION

0.5704975

1.7577232

0.00187901

HALLMARK_P53_PATHWAY

0.54956275

1.7134482

0.00262637

HALLMARK_APOPTOSIS

0.54781187

1.6755384

0.004013803

0.5385772

1.6667895

0.003868514

HALLMARK_INFLAMMATORY_RESPONSE

0.52368957

1.6260667

0.005695151

HALLMARK_IL6_JAK_STAT3_SIGNALING

0.55716753

1.579828

0.0013947

0.008902214

HALLMARK_INTERFERON_ALPHA_RESPONSE

0.55188423

1.5787069

0.008018492

0.5004975

1.5488061

0.009927

0.48502183

1.5044812

0.001242236

0.016797332

HALLMARK_ANGIOGENESIS

0.5948357

1.4922953

0.033383913

0.01766459

HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION

0.4732653

1.4622483

0.002528445

0.022271285

HALLMARK_UV_RESPONSE_DN

0.47187793

1.4172851

0.007692308

0.03291526

HALLMARK_BILE_ACID_METABOLISM

0.47735313

1.3960559

0.01899593

0.03932956

HALLMARK_COMPLEMENT

0.44702625

1.3827937

0.003911343

0.04317236

HALLMARK_ESTROGEN_RESPONSE_EARLY

0.42592376

1.3226221

0.011494253

0.080089346

0.410993

1.2768729

0.043209877

0.124076076

HALLMARK_INTERFERON_GAMMA_RESPONSE

HALLMARK_HEME_METABOLISM

HALLMARK_IL2_STAT5_SIGNALING

HALLMARK_KRAS_SIGNALING_UP

72

Table 3 Downregulation of Signaling Pathway Induced by PTC-028 in MDS-L Cells

NAME

ES

NES

NOM p-val

FDR q-val

HALLMARK_MYC_TARGETS_V1

-0.5312721

-1.8584391

0.004333333

HALLMARK_E2F_TARGETS

0.47660512

-1.6709709

0.005758667

HALLMARK_OXIDATIVE_PHOSPHORYLATION

-0.4207183

-1.5008153

0.029381553

73

Table 4 Upregulation of Signaling Pathway Induced by PTC-028 in SKM-1 Cells

NAME

ES

HALLMARK_TNFA_SIGNALING_VIA_NFKB

NES

NOM p-val

FDR q-val

0.6467109

2.1331067

HALLMARK_P53_PATHWAY

0.58609194

1.9506716

HALLMARK_APICAL_JUNCTION

0.58014596

1.9302019

HALLMARK_INFLAMMATORY_RESPONSE

0.55264056

1.8320497

2.87E-04

0.5546269

1.7967594

2.29E-04

HALLMARK_COAGULATION

0.55692613

1.7888622

1.91E-04

HALLMARK_MYOGENESIS

0.5327498

1.7650108

1.64E-04

HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION

0.5172625

1.7131113

5.57E-04

HALLMARK_TGF_BETA_SIGNALING

0.5863241

1.6650617

0.002022102

HALLMARK_IL6_JAK_STAT3_SIGNALING

0.5481379

1.661659

0.001819892

0.49732518

1.6503032

0.002014123

0.6159639

1.5805967

0.014059754

0.005915025

0.47112146

1.5666751

0.00652423

0.6000284

1.5502645

0.014876033

0.00710283

HALLMARK_UV_RESPONSE_UP

0.47220153

1.522929

0.008662837

HALLMARK_IL2_STAT5_SIGNALING

0.45630616

1.5213256

0.008390245

HALLMARK_KRAS_SIGNALING_UP

0.45659617

1.5073739

0.001416431

0.009566529

0.4333904

1.4450694

0.017967768

HALLMARK_INTERFERON_GAMMA_RESPONSE

0.43104485

1.4400431

0.001369863

0.018887723

HALLMARK_COMPLEMENT

0.43133524

1.4359385

0.005479452

0.018809563

0.5228781

1.4232961

0.031045752

0.021028811

HALLMARK_APOPTOSIS

HALLMARK_HYPOXIA

HALLMARK_NOTCH_SIGNALING

HALLMARK_ALLOGRAFT_REJECTION

HALLMARK_ANGIOGENESIS

HALLMARK_XENOBIOTIC_METABOLISM

HALLMARK_REACTIVE_OXIGEN_SPECIES_PATHWAY

74

Table 5 Downregulation of Signaling Pathway Induced by PTC-028 in SKM-1 Cells

NAME

ES

NES

HALLMARK_MYC_TARGETS_V1

0.53554523

-1.9443746

HALLMARK_E2F_TARGETS

0.42601994

-1.5342134

0.022642275

HALLMARK_PROTEIN_SECRETION

0.46719083

-1.5294216

0.002890173

0.01509485

HALLMARK_ANDROGEN_RESPONSE

0.40367532

-1.3367312

0.017045455

0.07223807

75

NOM p-val

FDR q-val

...