In Vitro Binding Affinity of fb-PMT with the thyrointegrin αvβ3.
In binding affinity experiments of fb-PMT, we confirmed that fb-PMT has a high affinity for the thyrointegrin αvβ3 receptors with a lower IC50 (50% inhibitory concentration) of 0.23 nM (Fig. 1).
Preclinical in vivo therapy experiments revealed potent fb-PMT anticancer activities.
Effects of fb-PMT therapy on K562-Luc human leukemic cell line FLT3-ITD primary human AML cells engrafted in transgenic mice.
In the K562-Luc engrafted in transgenic mice, blast cells appeared in the blood smears of NSG-S mice 10 days after engraftment, with an average value of 40%. After 21 days of fb-PMT daily s.c. injection, blast cell counts continually and consistently decreased in a dose-dependent manner in treated versus control groups, while animals in the control group showed increased blast cells in the peripheral blood. No blast cells could be detected in fb-PMT-treated animals (10 mg/kg) at the end of the treatment. Furthermore, there was no rebound increase in peripheral blast cells at 1 and 3 mg/kg with full sustained remission at fb-PMT dose of 10 mg/kg at 1–2 weeks post-discontinuation of treatment (Fig. 2, Supplementary Figure S1A).
On the other hand, primary AML cells (6373-FLT3-ITD) cells appeared in the blood smears of NSG-S mice 40 days after engraftment, with an average value of 26%. After 28 days of daily s.c. treatment, peripheral smears of treated animals were entirely normal at fb-PMT dose of 10 mg/kg. Daily s.c. injections of fb-PMT at 1, 3, and 10 mg/kg doses prevented blast cell expression/reproduction compared to controls by 54%, 75%, and 90.5%, respectively (Fig. 2 and Supplemental Figure S1B).
After termination, the bone marrow K562-Luc engrafted mice with a daily treatment of fb-PMT at 3 mg/kg manifested 30–40% infiltration with blast cells, while 70% maturation could be detected. fb-PMT-treated animals at 10 mg/kg dose presented bone marrows with blast cell counts < 5% and > 95%, normal maturation have been documented (segmented neutrophils). The remission was maintained in all treated mice at least 2 weeks after fb-PMT therapy discontinuation (Fig. 3A, 3B and Supplemental Figure S2A).
IVIS scans and histopathological results at sacrifice showed a dose-dependent decrease of brain, lung, liver, and spleen infiltration with the leukemic cells in the group of fb-PMT-treated mice (10 mg/kg) in comparison to control group Supplemental Figure S3. The fb-PMT therapy at 10 mg/kg dose in the ON + OFF treatment group resulted in successfully maintained remission in all animals 2 weeks after withdrawal of the daily treatment. The sustained remission was confirmed using blood smear analyses, IVIS scans, flowcytometry and histopathological examinations.
Regarding the bone marrow samples from mice engrafted with primary AML cells (FLT3-ITD), the fb-PMT-treated group (10 mg/kg) restored the normal bone marrow maturation with abundant megakaryocytes in comparison to control animals (Fig. 3C and 3D). The results were confirmed with flowcytometry and immunohistochemistry analysis (Supplemental Figure S2)
Furthermore, we evaluated the splenic infiltration in our animal models. Histopathological results showed a marked decrease of splenic metastases of the leukemic cells in the group treated with fb-PMT (10 mg/kg) compared to control group (Fig. 4). Similarly, to the K562-Luc AML experiments, the primary AML model (ON + OFF) group (10 mg/kg) manifested the successful maintenance of remission 2 weeks after withdrawal of daily therapy. The splenic weight showed marked decrease (80%) even with the low dose (1 mg/kg). The ON + OFF groups maintained normal splenic weight in comparison to control, which may reflect successful prevention of engraftment (Fig. 4A). Treatment with fb-PMT daily for 28 days (ON treatment) or 28 days ON and 14 days off treatment (ON + OFF treatment) resulted in maximal suppression of primary AML infiltration into the spleen of AML engrafted transgenic mice (Fig. 4B).
Microarray analyses of fb-PMT effects on gene expression in human AML cells
Previous contributions have established that one of the principal molecular mechanisms of anticancer activities of thyrointegrin αvβ3 antagonists is exemplified by significant changes in expression of genes essential for growth and survival of malignant cells [28–32]. Therefore, to gain insights into mechanisms of the fb-PMT activities against cancer cells, it was of interest to do genome-wide gene expression profiling experiments. Following 2 days of a single-dose treatment with 30 µM of fb-PMT, RNA samples were extracted from biological replicates of control and treated human AML cells and subjected to microarray analyses (Methods). Results of these analyses are reported in Figs. 5–7 and Tables 1–3.
Table 1
Examples of mimicry of pathway activation and pathway interference gene expression signatures (GES) identified in fb-PMT-treated K562 cells.
fb-PMT pathway activation GES
|
Genes
|
P-value*
|
Odds Ratio*
|
Combined Score*
|
RB1 pathway signature (n = 10)
|
STARD4;TMEM2;SESN3;SPIN4;SLX4IP;CD109;CENPE;CEP135;TCP11L2;PLD1
|
3.70E-04
|
6.266319
|
49.51741
|
IRF9 pathway signature (n = 6)
|
CENPE;EID3;SESN3;CD109;ASB7;TCP11L2
|
5.12E-04
|
7.54717
|
57.18613
|
MAML1 pathway signature (n = 6)
|
CENPE;PTAR1;STARD4;TMEM30A;CD109;TCP11L2
|
0.001655
|
4.696673
|
30.07697
|
RAP1A pathway signature (n = 5)
|
CENPE;SPIN4;ACSL1;ELOVL7;IL18R1
|
0.001067
|
6.410256
|
43.86473
|
GATA4 pathway signature (n = 5)
|
ERRFI1;ACSL1;LRRC28;CD9;ELOVL7
|
4.15E-04
|
7.905138
|
61.56706
|
fb-PMT pathway interference GES
|
Genes
|
P-value*
|
Odds Ratio*
|
Combined Score*
|
MYC pathway signature (n = 8)
|
ERRFI1;CENPE;BLOC1S6;TMEM30A;CD109;SESN3;ZNF17;KDSR
|
2.04E-04
|
9.21659
|
78.29477
|
HIF1A pathway signature (n = 6)
|
ERRFI1;ERV3-1;TCP11L2;SH3BGRL2;ELOVL7;IL18R1
|
1.48E-04
|
7.430341
|
65.52441
|
TWIST1 pathway signature (n-5)
|
EID3;SESN3;CD109;TCP11L2;ASB7
|
0.00152
|
5.91716
|
38.39501
|
TFAP2C pathway signature (n = 5)
|
CENPE;SPIN4;ACSL1;CD109;ELOVL7
|
5.21E-04
|
7.518797
|
56.84253
|
Legend: GES were identified based on the analyses of top 50 down-regulated genes in fb-PMT-treated K562 cells; *, Statistical metrics were defined by the Enrichr bioinformatics platform (Methods). |
Table 2
Mimicry of pathway activation and pathway interference gene expression signatures (GES) identified in fb-PMT-treated K562 cells.
Enriched Terms
|
P-value*
|
Adjusted P-value*
|
Odds Ratio*
|
Combined Score*
|
Genes
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1659 DOWN
|
2.89E-07
|
5.65E-04
|
31.44654
|
473.5
|
CENPE;EID3;SESN3;CD109;ASB7
|
TFAP2C SIRNA HUMAN GSE15481 CREEDSID GENE 2895 DOWN
|
2.94E-07
|
2.88E-04
|
31.32832
|
471.2
|
CENPE;ACSL1;SPIN4;CD109;ELOVL7
|
TFAP2C KD HUMAN GSE15481 CREEDSID GENE 2970 DOWN
|
3.60E-07
|
2.35E-04
|
30.08424
|
446.4
|
CENPE;ACSL1;SPIN4;CD109;ELOVL7
|
TWIST1 OE MOUSE GSE50002 CREEDSID GENE 1075 UP
|
9.62E-07
|
4.71E-04
|
24.65483
|
341.6
|
EID3;SESN3;CD109;ASB7;TCP11L2
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1663 DOWN
|
1.16E-06
|
4.54E-04
|
23.74169
|
324.5
|
EID3;SESN3;CD109;ASB7;TCP11L2
|
MYC OE U2OS HUMAN GSE59819 RNASEQ UP
|
6.23E-06
|
0.002034
|
30.72197
|
368.2
|
CENPE;ERRFI1;TMEM30A;CD109
|
MYC OE U2OS HUMAN GSE66789 RNASEQ UP
|
6.23E-06
|
0.001743
|
30.72197
|
368.2
|
CENPE;ERRFI1;TMEM30A;CD109
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1656 DOWN
|
8.26E-06
|
0.002021
|
28.6123
|
334.9
|
SESN3;CD109;ASB7;TCP11L2
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1653 DOWN
|
1.43E-05
|
0.00312
|
24.87562
|
277.4
|
SESN3;CD109;ASB7;TCP11L2
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1657 DOWN
|
2.20E-05
|
0.004317
|
22.29654
|
239.1
|
SESN3;CD109;ASB7;TCP11L2
|
IRF9 OE HUMAN GSE50002 CREEDSID GENE 1654 DOWN
|
2.45E-05
|
0.004353
|
21.71553
|
230.6
|
SESN3;CD109;ASB7;TCP11L2
|
HIF1A KO MOUSE GSE35111 CREEDSID GENE 1406 DOWN
|
2.98E-05
|
0.004868
|
20.63983
|
215.1
|
ERRFI1;TCP11L2;ELOVL7;IL18R1
|
Legend: GES were identified based on the analyses of 12 down-regulated genes in fb-PMT-treated K562 cells; *, Statistical metrics were defined by the Enrichr bioinformatics platform (Methods). |
Table 3
Examples of functionally-significant genes down-regulated by the fb-PMT treatment in KG1A human AML cells.
Description
|
Gene Symbol
|
Fold Change
|
P-value
|
X-linked inhibitor of apoptosis, E3 ubiquitin protein ligase
|
XIAP
|
-2.9
|
0.0185
|
osteoclast stimulating factor 1
|
OSTF1
|
-2.86
|
0.0331
|
signal transducer and activator of transcription 2
|
STAT2
|
-2.81
|
0.0202
|
thymopoietin
|
TMPO
|
-2.59
|
0.0069
|
signal transducer and activator of transcription 4
|
STAT4
|
-2.54
|
0.0451
|
YES proto-oncogene 1, Src family tyrosine kinase
|
YES1
|
-2.49
|
0.0315
|
polymerase (RNA) II (DNA directed) polypeptide B, 140kDa
|
POLR2B
|
-2.44
|
0.0354
|
cyclin-dependent kinase 14
|
CDK14
|
-2.21
|
0.0402
|
TTK protein kinase
|
TTK
|
-2.2
|
0.046
|
topoisomerase (DNA) III alpha
|
TOP3A
|
-2.12
|
0.0116
|
prohibitin 2; small Cajal body-specific RNA 12
|
PHB2
|
-2.03
|
0.042
|
cyclin-dependent kinase 17
|
CDK17
|
-1.97
|
0.0303
|
phosphoglycerate kinase 2
|
PGK2
|
-1.85
|
0.0471
|
polymerase (DNA directed), epsilon 2, accessory subunit
|
POLE2
|
-1.83
|
0.0441
|
B-Raf proto-oncogene, serine/threonine kinase
|
BRAF
|
-1.81
|
0.0315
|
growth factor receptor bound protein 2
|
GRB2
|
-1.81
|
0.0301
|
Pim-1 proto-oncogene, serine/threonine kinase
|
PIM1
|
-1.71
|
0.038
|
serine/threonine kinase 4
|
STK4
|
-1.69
|
0.0135
|
prostaglandin E synthase 3 (cytosolic)
|
PTGES3
|
-1.56
|
0.0429
|
B-cell CLL/lymphoma 9
|
BCL9
|
-1.52
|
0.0334
|
Overall, there were 518 significantly down-regulated gene expression records and 283 significantly up-regulated gene expression records, expression of which were changed at least 1.5-fold in fb-PMT-treated K562 cells. In fb-PMT-treated KG1a cells, 223 significantly down-regulated gene expression records and 191 significantly up-regulated gene expression records were identified, expression of which were changed at least 1.5-fold in fb-PMT-treated cells. All DEG identified by the Affymetrix Expression Console software were subjected to GSEA employing a panel of 29 genomic databases (Methods). Analyses of fb-PMT treatment-induced DEGs in both K562 and KG1a cells identified sets of down-regulated genes and a genomic database of Transcription Factor (TF) Perturbations Followed by Expression as the most informative setting among comparison records. GSEA of down-regulated DEGs using the TF Perturbations Followed by Expression database identified 60 and 84 significantly enriched records (adjusted p value < 0.05) for fb-PMT-treated K562 and KG1a cells, respectively.
GSEA of fb-PMT effects on gene expression revealed signatures of the molecular mimicry of both activation of and interference with multiple transcriptional pathways:
Follow-up analyses of down-regulated DEGs using the TF Perturbations Followed by Expression database identified multiple examples of the molecular mimicry of pathway activation and pathway interference gene expression signatures (GES) identified in fb-PMT-treated human AML cells (Table 1; Fig. 5). Notable examples of the fb-PMT-induced GES of transcriptional pathway’s activation include RB1, IRF9, MAML1, RAP1A, and GATA4 pathways, and examples of the fb-PMT-induced GES of pathway’s interference include MYC, HIF1A, TWIST1, and TFAP2C pathways. Integrations of DEGs comprising fb-PMT-induced GES listed in Table 1 identified a total of 25 genes, differential expression of which appears to define molecular signals of either activation of or interference with transcriptional pathways in fb-PMT-treated human AML cells (Fig. 5). GSEA of genes comprising the 25-gene and 12-gene expression signatures validated their significance in defining observations of the molecular mimicry of transcriptional pathways’ activation and interference induced by fb-PMT treatment in human AML cells (Figs. 5–7, Table 2). GSEA of all significant DEGs confirmed and extended these findings.
Interestingly, GSEA identified the SNAI transcriptional pathway as the most significantly enriched pathway of the molecular interference observed in K562 cells treated with fb-PMT among either down-regulated or up-regulated DEGs (Supplemental Fig. 4). Additional examples of the specific genes and pathways of potential functional significance revealed by the GSEA of 233 genes down-regulated in KG1a cells after fb-PMT treatment are shown in Table 3. Of note, GSEA of the LINCS L1000 Ligand Perturbations database of up-regulated genes revealed evidence of molecular interference with functions of multiple growth factors in human cancer cell lines (Fig. 7).
GSEA of fb-PMT effects on gene expression revealed signatures of the molecular interference with a regulatory crosstalk of estrogen pathway and multi-kinase transcriptional matrix of cell cycle progression.
GSEA of databases of Ligand perturbations from GEO focused on up-regulated genes and Ligand perturbations from GEO focused on down-regulated genes revealed multiple examples of molecular interference with transcriptional signaling induced by many endogenous ligands, among which the enrichment of genes implicated in estrogen signaling was particularly apparent (Fig. 6 and Supplemental Fig. 4). Similarly, GSEA of the ARCHS4 Kinases Co-expression database revealed evidence of the molecular interference with functions of multiple kinases. GSEA of the integrated 69-gene signature of the fb-PMT interference with estrogen signaling in human AML cells identified 16 genes that appear implicated in transcriptional regulatory crosstalk of estrogens with multiple kinases in human tissues (Supplemental Fig. 5). Integration of 16 estrogen-regulated genes with 35 genes encoding kinases engaged in regulatory crosstalk in human tissues generated the 50-gene signature of estrogen pathway/multi-kinase gene expression regulatory matrix, which appears engaged in the cell cycle progression pathway (Supplemental Fig. 5). Intriguingly, GSEA of the 50-gene interference signature with estrogen-kinase regulatory matrix using the DisGeNET database of human disorders revealed that these genes were implicated in a remarkably broad spectrum of human malignancies (Supplemental Fig. 5),