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Posts Tagged ‘FLT3 gene on CHromosome 13’

miRNA Therapeutic Promise

Curator: Larry H. Bernstein, MD, FCAP

 

MicroRNA Expression Could Be Key to Leukemia Treatment

http://www.genengnews.com/gen-news-highlights/microrna-expression-could-be-key-to-leukemia-treatment/81252662/

MicroRNA Expression Could Be Key to Leukemia Treatment

Generalized gene regulation mechanisms of miRNAs. [NIH]

 

Increasingly, cancer researchers are discovering novel biological pathways that regulate the expression of various genes that are often strongly associated with tumorigenesis. These new molecular mechanisms represent important potential therapeutic targets for aggressive and difficult-to-treat cancers. In particular, microRNAs (miRNAs)—small, noncoding genetic material that regulates gene expression—have steadily become implicated in the progression of some cancers.

Now, researchers at the University of Cincinnati (UC) have found a particular signaling route for a microRNA, miR-22, that they believe leads to targets for acute myeloid leukemia (AML), the most common type of fast-growing cancer of the blood and bone marrow.

The findings from this study were published recently in Nature Communications in an article entitled “miR-22 Has a Potent Anti-Tumour Role with Therapeutic Potential in Acute Myeloid Leukaemia.”

Structure of mi-22 miccroRNA. [Ppgardne at el., via Wikimedia Commons]

Increasingly, cancer researchers are discovering novel biological pathways that regulate the expression of various genes that are often strongly associated with tumorigenesis. These new molecular mechanisms represent important potential therapeutic targets for aggressive and difficult-to-treat cancers. In particular, microRNAs (miRNAs)—small, noncoding genetic material that regulates gene expression—have steadily become implicated in the progression of some cancers.

Now, researchers at the University of Cincinnati (UC) have found a particular signaling route for a microRNA, miR-22, that they believe leads to targets for acute myeloid leukemia (AML), the most common type of fast-growing cancer of the blood and bone marrow.

The findings from this study were published recently in Nature Communications in an article entitled “miR-22 Has a Potent Anti-Tumour Role with Therapeutic Potential in Acute Myeloid Leukaemia.”

“MicroRNAs make up a class of small, noncoding internal RNAs that control a gene’s job, or expression, by directing their target messaging RNAs, or mRNAs, to inhibit or stop. Cellular organisms use mRNA to convey genetic information,” explained senior study author Jianjun Chen, Ph.D., associate professor in the department of cancer biology at the UC College of Medicine. “Previous research has shown that microRNA miR-22 is linked to breast cancer and other blood disorders which sometimes turn into AML, but we found in this study that it could be an essential anti-tumor gatekeeper in AML when it is down-regulated, meaning its function is minimized.”

AML—most common type of acute leukemia—arises when the bone marrow begins to make blasts, cells that have not yet completely matured. These blast cells typically develop into white blood cells; however, in AML the cells do not develop and are unable to aid in warding off infections. In the current study, the UC team describes how altering the expression of miR-22 affected AML pathogenesis.

“When we forced miR-22 expression, we saw difficulty in leukemia cells developing, growing, and thriving. miR-22 targets multiple cancer-causing genes (CRTC1, FLT3, and MYCBP) and blocks certain pathways (CREB and MYC),” Dr. Chen noted. “The downregulation, or decreased output, of miR-22 in AML, is caused by the loss of the number of DNA being copied and/or stopping their expression through a pathway called TET1/GFI1/EZH2/SIN3A. Also, nanoparticles carrying miR-22 DNA oligonucleotides (short nucleic acid molecules) prevented leukemia advancement.”

The investigators conducted the study using bone marrow transplant samples and animal models. The researchers showed that the ten-eleven translocation proteins (TET1/2/3) in mammals helped to control genetic expression in normal developmental processes. This was in sharp contrast to mutations that cause function loss and tumor-slowing with TET2, which has been observed previously in blood and stem cell cancers.

“We recently reported that TET1 plays an essential cancer generating role in certain AML where it activates expression of homeobox genes, which are a large family of similar genes that direct the formation of many body structures during early embryonic development,” remarked Dr. Chen. “However, it is unknown whether TET1 can also function as a repressor for cellular function in cancer, and its role in microRNA expression has rarely been studied.”

Dr. Chen added that these findings are important in targeting a cancer that is both common and fatal, stating that “the majority of patients with ALM usually don’t survive longer than 5 years, even with chemotherapy, which is why the development of new effective therapies based on the underlying mechanisms of the disease is so important.”

“Our study uncovers a previously unappreciated signaling pathway (TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC) and provides new insights into genetic mechanisms causing and progressing AML and also highlights the clinical potential of miR-22-based AML therapy. More research on this pathway and ways to target it are necessary,” Dr. Chen concluded.

 

miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia

Xi JiangChao HuStephen ArnovitzJason BugnoMiao YuZhixiang ZuoPing Chen, et al.
Nature Communications 26 Apr 2016; 7(11452).    http://dx.doi.org:/doi:10.1038/ncomms11452

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy.

 

As one of the most common and fatal forms of hematopoietic malignancies, acute myeloid leukaemia (AML) is frequently associated with diverse chromosome translocations (for example t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA and t(8;21)/AML1-ETO) and molecular abnormalities (for example, internal tandem duplications of FLT3 (FLT3-ITD) and mutations in nucleophosmin (NPM1c+))1. Despite intensive chemotherapies, the majority of patients with AML fail to survive longer than 5 years2, 3. Thus, development of effective therapeutic strategies based on a better understanding of the molecular mechanisms underlying the pathogenesis of AML is urgently needed.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that post-transcriptionally regulate gene expression4. Individual miRNAs may play distinct roles in cancers originating from different tissues or even from different lineages of hematopoietic cells4. It is unclear whether a single miRNA can play distinct roles between malignancies originating from the same hematopoietic lineage, such as de novo AML and myelodysplastic syndrome (MDS). Although around 30% of MDS cases transform to AML, the genetic and epigenetic landscapes of MDS or MDS-derived AML are largely different from those of de novo AML5, 6. MDS and MDS-derived AML are more responsive to hypomethylating agents than de novo AML7. The molecular mechanisms underlying the distinct pathogenesis and drug response between MDS (or MDS-derived AML) and de novo AML remain unclear.

The ten-eleven translocation (Tet1/2/3) proteins play critical transcriptional regulatory roles in normal developmental processes as activators or repressors8, 9, 10. In contrast to the frequent loss-of-function mutations and tumour-suppressor role of TET2 observed in hematopoietic malignancies11, 12, 13, we recently reported that TET1 plays an essential oncogenic role in MLL-rearranged AML where it activates expression of homeobox genes14. However, it is unknown whether TET1 can also function as a transcriptional repressor in cancer. Moreover, Tet1-mediated regulation of miRNA expression has rarely been studied10.

In the present study, we demonstrate that miR-22, an oncogenic miRNA reported in breast cancer and MDS15, 16, is significantly downregulated in most cases of de novo AML due to TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. miR-22 functions as an essential anti-tumour gatekeeper in various AML and holds great therapeutic potential to treat AML.

 

The downregulation of miR-22 in de novo AML

Through Exiqon miRNA array profiling, we previously identified a set of miRNAs, such as miR-150, miR-148a, miR-29a, miR-29b, miR-184, miR-342, miR-423 and miR-22, which are significantly downregulated in AML compared with normal controls17. Here we showed that among all the above miRNAs, miR-150 and especially miR-22 exhibited the most significant and consistent inhibitory effect on MLL-AF9-induced cell immortalization in colony-forming/replating assays (CFA) (Supplementary Fig. 1a). In contrast to the reported upregulation of miR-22 in MDS16, our original microarray data17 (Fig. 1a,b) and new quantitative PCR-independent validation data (Supplementary Fig. 1b) demonstrated a significant and global downregulation of miR-22 in de novo AML relative to normal controls. Notably, miR-22 is significantly downregulated in AML samples (P<0.05) compared with all three sub-populations of normal control cells, that is, normal CD34+ hematopoietic stem/progenitor cells (HSPCs), CD33+ myeloid progenitor cells, or mononuclear cells (MNCs) (Fig. 1a). Expression of miR-22 is significantly downregulated in all or the majority of individual subsets of AML samples than in the normal CD33+ or CD34+ cell samples (Fig. 1b).

Figure 1: miR-22 inhibits AML cell transformation and leukemogenesis.

miR-22 inhibits AML cell transformation and leukemogenesis.

(a,b) Exiqon microRNA profiling assay showed that miR-22 is significantly (P<0.05) downregulated in the entire set of AML set (n=85) (a) or each individual subset (b), relative to normal controls. The expression data were log(2) transformed and mean-centred. Mean±s.e.m. values were shown. (c) Comparison of effects of in-house miR-22, miR-22_Song16 and miR-22 mutant (miR-22mut; see the mutation sequence at the top) on MLL-AF9-induced colony forming. CFAs were performed using mouse BM progenitor (Lin) cells transduced with MSCV-neo+MSCV-PIG (Ctrl), MSCV-neo-MLL-AF9+MSCV-PIG (MLL-AF9), or MSCV-neo-MLL-AF9+MSCV-PIG-miR-22/miR-22_Song/miR-22mut. (d) Effects of miR-22 on the colony forming induced by multiple fusion genes. CFA was performed using wild-type BM progenitor cells co-transduced with MSCV-neo-MLL-AF9 (MA9), -MLL-AF10 (MA10), -PML-RARA (PR) or –AML1-ETO9a(AE9a)19, together with MSCV-PIG (Ctrl) or MSCV-PIG-miR-22 (+miR-22), as well as miR-22−/− BM progenitors co-transduced with individual fusion genes and MSCV-PIG. Colony counts (mean±s.d.) of the second round of plating are shown. *P<0.05; **P<0.01. (e,f) Effect of miR-22 on MLL-AF9-induced primary leukemogenesis. Kaplan–Meier curves are shown for six cohorts of transplanted mice including MSCVneo+MSCV-PIG (Ctrl; n=5), MSCVneo+MSCV-PIG-miR-22 (miR-22; n=5), MSCVneo-MLL-AF9+MSCV-PIG (MA9; n=8), MSCVneo-MLL-AF9+MSCV-PIG-miR-150 (MA9+miR-150, n=6), MSCVneo-MLL-AF9+MSCV-PIG-miR-22 (MA9+miR-22; n=10) and MSCVneo-MLL-AF9+MSCV-PIG-miR-22mutant (MA9+miR-22mut; n=5) (e); Wright–Giemsa stained PB and bone marrow (BM), and hematoxylin and eosin (H&E) stained spleen and liver of the primary BMT recipient mice at the end point are shown (f). (g) Effect of miR-22 on MLL-AF10-induced primary leukemogenesis. Kaplan–Meier curves are shown for two cohorts of transplanted mice including MSCVneo-MLL-AF10+MSCV-PIG (MA10; n=5) and MSCVneo-MLL-AF10+MSCV-PIG-miR-22 (MA10+miR-22; n=5). (h) miR-22 knockout promotes AE9a-induced leukemogenesis. Kaplan–Meier curves are shown for mice transplanted with wild-type or miR-22−/− BM progenitor cells transduced MSCV-PIG-AE9a (n=5 for each group). The P values were generated by t-test (ad) or log-rank test (e,g,h).

To rule out the possibility that the inhibitory effect of miR-22 shown in Supplementary Fig. 1a was due to a non-specific effect of our miR-22 construct, we included the MSCV-PIG-miR-22 construct from Song et al.16 in a repeated CFA. Both miR-22 constructs dramatically inhibited MLL-AF9-induced colony formation (Fig. 1c). As the ‘seed’ sequences at the 5′ end of individual miRNAs are essential for the miRNA-target binding18, we also mutated the 6-bases ‘seed’ sequence of miR-22 and found that the miR-22 mutant did not inhibit colony formation anymore (Fig. 1c). In human AML cells, forced expression of miR-22, but not miR-22 mutant, significantly inhibited cell viability and growth/proliferation, while promoting apoptosis (Supplementary Fig. 1c,d).

Furthermore, as miR-22 is globally downregulated in all major types of AML (Fig. 1b), we also investigated the role of miR-22 in colony formation induced by other oncogenic fusion genes, including MLL-AF10/t(10;11), PML-RARA/t(15;17) and AML1-ETO9a/t(8;21) (ref. 19). As expected, forced expression of miR-22 significantly inhibited colony formation induced by all individual oncogenic fusions; conversely, miR-22 knockout20 significantly enhanced colony forming (Fig. 1d). These results suggest that miR-22 likely plays a broad anti-tumour role in AML.

In accordance with the potential anti-tumour function of miR-22 in AML, miR-22 was expressed at a significantly higher level (P<0.05) in human normal CD33+ myeloid progenitor cells than in more immature CD34+ HSPCs or MNC cells (a mixed population containing both primitive progenitors and committed cells) (Fig. 1a,b), implying that miR-22 is upregulated during normal myelopoiesis. Similarly, we showed that miR-22 was also expressed at a significantly higher level in mouse normal bone marrow (BM) myeloid (Gr-1+/Mac-1+) cells, relative to lineage negative (Lin) progenitor cells, long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and committed progenitors (CPs) (Supplementary Fig. 1e), further suggesting that miR-22 is upregulated in normal myelopoiesis.

The anti-tumour effect of miR-22 in the pathogenesis of AML

Through bone marrow transplantation (BMT) assays, we showed that forced expression of miR-22 (but not miR-22 mutant) dramatically blocked MLL-AF9 (MA9)-mediated leukemogenesis in primary BMT recipient mice, with a more potent inhibitory effect than miR-150 (Fig. 1e;Supplementary Fig. 2a). All MA9+miR-22 mice exhibited normal morphologies in peripheral blood (PB), BM, spleen and liver tissues (Fig. 1f), with a substantially reduced c-Kit+ blast cell population in BM (Supplementary Fig. 2b). Forced expression of miR-22 also almost completely inhibited leukemogenesis induced by MLL-AF10 (Fig. 1g; Supplementary Fig. 2a). Conversely, miR-22 knockout significantly promoted AML1-ETO9a (AE9a)-induced AML (Fig. 1h). Thus, the repression of miR-22 is critical for the development of primary AML. Notably, forced expression of miR-22 inMLL-AF9 and MLL-AF10 leukaemia mouse models caused only a 2–3-fold increase in miR-22 expression level (Supplementary Fig. 2a), in a degree comparable to the difference in miR-22 expression levels between human AML samples and normal controls (Fig. 1a), suggesting that a 2–3-fold change in miR-22 expression level appears to be able to exert significant physiological or pathological effects.

To examine whether the maintenance of AML is also dependent on the repression of miR-22, we performed secondary BMT assays. Forced expression of miR-22 remarkably inhibited progression of MLL-AF9-, AE9a– or FLT3-ITD/NPM1c+-induced AML in secondary recipient mice (Fig. 2a–d), resulting in largely normal morphologies in PB, BM, spleen and liver tissues (Fig. 2b;Supplementary Fig. 2c). Collectively, our findings demonstrate that miR-22 is a pivotal anti-tumour gatekeeper in both development and maintenance of various AML.

Figure 2: Effect of miR-22 on the maintenance of AML in vivo.

Effect of miR-22 on the maintenance of AML in vivo.

(a,b) Effect of miR-22 on the maintenance of MLL-AF9-induced AML in secondary BMT recipient mice. The secondary BMT recipients were transplanted with BM blast cells from the primary MLL-AF9 AML mice retrovirally transduced with MSCV-PIG+MSCVneo (MA9-AML+Ctrl; n=7) or MSCV-PIG+MSCVneo-miR-22 (MA9-AML+miR-22; n=10). Kaplan–Meier curves (a) and Wright–Giemsa or H&E-stained PB, BM, spleen and liver (b) of the secondary leukaemic mice are shown. (c,d) Effect of miR-22 on the maintenance/progression of AML1-ETO9a (AE9a)-induced AML (c) or FLT3-ITD/NPM1c+-induced AML (d) in secondary BMT recipient mice (n=5 for each group). Kaplan–Meier curves and P values (log-rank test) are shown.

 

Identification of critical target genes of miR-22 in AML

To identify potential targets of miR-22 in AML, we performed a series of data analysis. Analysis of In-house_81S (ref. 21) and TCGA_177S (ref. 22) data sets revealed a total of 999 genes exhibiting significant inverse correlations with miR-22 in expression. Of them, 137 genes, including 21 potential targets of miR-22 as predicted by TargetScan18 (Supplementary Table 1), were significantly upregulated in both human and mouse AML compared with normal controls as detected in two additional in-house data sets14, 23. Among the 21 potential targets, CRTC1, ETV6and FLT3 are known oncogenes24, 25, 26, 27, 28, 29. We then focused on these three genes, along with MYCBP that encodes the MYC-binding protein and is an experimentally validated target of miR-22 (ref. 30) although due to a technical issue it was not shown in the 21-gene list (Supplementary Table 1), for further studies.

As expected, all four genes were significantly downregulated in expression by ectopic expression of miR-22 in human MONOMAC-6/t(9;11) cells (Fig. 3a). The coincidence of downregulation of those genes and upregulation of miR-22 was also observed in mouse MLL-ENL-ERtm cells, a leukaemic cell line with an inducible MLL-ENL derivative31, when MLL-ENL was depleted by 4-hydroxy-tamoxifen (4-OHT) withdrawal (Fig. 3b; Supplementary Fig. 3a). While MLL-AF9 remarkably promoted expression of those four genes in mouse BM progenitor cells, co-expressed miR-22 reversed the upregulation (Fig. 3c). In leukaemia BM blast cells of mice with MLL-AF9-induced AML, the expression of Crtc1, Flt3 and Mycbp, but not Etv6, was significantly downregulated by co-expressed miR-22 (but not by miR-22 mutant) (Fig. 3d). Because miR-22-mediated downregulation of Etv6 could be observed only in the in vitro models (Fig. 3a–c), but not in the in vivo model (Fig. 3d), which was probably due to the difference between in vitro and in vivo microenvironments, we decided to focus on the three target genes (that is, Crtc1, Flt3 and Mycbp) that showed consistent patterns between in vitro and in vivo for further studies. The repression of Crtc1, Flt3 and Mycbpwas also found in leukaemia BM cells of mice with AE9a or FLT3-ITD/NPM1c+-induced AML (Fig. 3e,f). As Mycbp is already a known target of miR-22 (ref. 30), here we further confirmed that FLT3and CRTC1 are also direct targets of miR-22 (Fig. 3g,h). The downregulation of CRTC1, FLT3 and MYCBP by miR-22 at the protein level was confirmed in both human and mouse leukaemic cells (Supplementary Fig. 3b,c). Overexpression of miR-22 had no significant influence on the level of leukaemia fusion genes (Supplementary Fig. 3d).

Figure 3: miR-22 targets multiple oncogenes.

miR-22 targets multiple oncogenes.

(a) Downregulation of CRTC1, FLT3, MYCBP and ETV6 by forced expression of miR-22 in MONOMAC-6 cells. Expression of these genes was detected 48h post transfection of MSCV-PIG (Ctrl) or MSCV-PIG-miR-22 (miR-22). (b) Crtc1, Flt3, Mycbp and Etv6 levels in MLL-ENL-ERtm cells after withdrawal of 4-OHT for 0, 7 or 10 days. (c) Expression levels of Crtc1, Flt3, Mycbp and Etv6 in mouse BM progenitor cells retrovirally transduced with MSCV-PIG+MSCV-neo (Ctrl), MSCV-PIG-miR-22+MSCV-neo (miR-22), MSCV-PIG+MSCV-neo-MLL-AF9 (MLL-AF9) or MSCV-PIG-miR-22+MSCV-neo-MLL-AF9 (MLL-AF9+miR-22). (d) Expression levels of Crtc1, Flt3, Mycbp and Etv6 in BM blast cells of leukaemic mice transplanted with MLL-AF9, MLL-AF9+miR-22 or MLL-AF9+miR-22mut primary leukaemic cells. (e,f) Expression levels of Crtc1, Flt3 and Mycbp in BM blast cells of leukaemic mice transplanted with MSCV-PIG or MSCV-PIG-miR-22-retrovirally transduced AE9a (e) or FLT3-ITD/NPM1c+ (f) primary leukaemic cells. (g) Putative miR-22 target sites and mutants in the 3′UTRs of CRTC1 (upper panel) and FLT3(lower panel). (h) Effects of miR-22 on luciferase activity of the reporter gene bearing wild type or mutant 3′UTRs of CRTC1 or FLT3 in HEK293T cells. The mean±s.d. values from three replicates are shown.*P<0.05, t-test.

Co-expression of the coding region (CDS) of each of the three target genes (that is, CRTC1, FLT3and MYCBP) largely reversed the effects of miR-22 on cell viability, apoptosis and proliferation (Fig. 4a–e). More importantly, in vivo BMT assays showed that co-expressing CRTC1, FLT3 orMYCBP largely rescued the inhibitory effect of miR-22 on leukemogenesis (Fig. 4f,g;Supplementary Fig. 3e). Our data thus suggest that CRTC1, FLT3 and MYCBP are functionally important targets of miR-22 in AML.

Figure 4: Multiple onocgenes are functionally important targets of miR-22 in AML.

Multiple onocgenes are functionally important targets of miR-22 in AML.

(a,b) Relative viability (a) and apoptosis (b) levels of MONOMAC-6 cells transfected with MSCV-PIG-CRTC1, -FLT3 or –MYCBP alone, or together with MSCVneo-miR-22. Values were detected 48h post transfection. (c–e) Rescue effects of CRTC1 (c), FLT3 (d) and MYCBP (e) on the inhibition of MONOMAC-6 growth mediated by miR-22. Cell counts at the indicated time points are shown. Mean±s.d. values are shown. *P<0.05, t-test. (f) In vivo rescue effects of CRTC1, FLT3 and MYCBP on the inhibition of MLL-AF9-induced leukemogenesis mediated by miR-22. The secondary recipients were transplanted with BM blast cells of the primary MLL-AF9 leukaemic mice retrovirally transduced with MSCVneo+MSCV-PIG (MA9-AML+Ctrl; n=7), MSCVneo-miR-22+MSCV-PIG (MA9-AML+miR-22; n=10), MSCVneo-miR-22+MSCV-PIG-CRTC1 (MA9-AML+miR-22+CRTC1; n=5), MSCVneo-miR-22+MSCV-PIG-FLT3 (MA9-AML+miR-22+FLT3; n=6) or MSCVneo-miR-22+MSCV-PIG-MYCBP (MA9-AML+miR-22+MYCBP; n=6). Kaplan–Meier curves for all the five groups of transplanted mice are shown. MA9-AML+Ctrl versus MA9-AML+miR-22, P<0.001 (log-rank test); MA9-AML+Ctrl versus any other groups,P>0.05 (log-rank test). (g) Wright–Giemsa stained PB and BM, and H&E stained spleen and liver of the secondary leukaemic mice.

miR-22 represses both CREB and MYC signalling pathways

DNA copy-number loss of miR-22 gene locus in AML

Expression of miR-22 is epigenetically repressed in AML

 

Figure 5: Transcriptional correlation between miR-22 and TET1.

http://www.nature.com/ncomms/2016/160426/ncomms11452/images_article/ncomms11452-f5.jpg

(a) Correlation between the expression levels of miR-22 and TET1 in three independent AML patient databases. All expression data were log(2) transformed; the data in In-house_81S were also mean-centred. The correlation coefficient (r) and P values were detected by ‘Pearson Correlation’, and the correlation regression lines were drawn with the ‘linear regression’ algorithm. (b) Expression of pri-, pre- and mature miR-22, and Tet1/2/3 in colony-forming cells of wild-type mouse BM progenitors retrovirally transduced with MSCVneo (Ctrl), MSCVneo-MLL-AF9 (MLL-AF9), MSCVneo-MLL-AF10 (MLL-AF10) or MSCVneo-AE9a (AE9a), or of FLT3-ITD/NPM1c+ mouse BM progenitors transduced with MSCVneo (FLT3-ITD+/NPM1c+). (c) Expression of miR-22 and Tet1/2/3 in MLL-ENL-ERtm cells. Expression levels were detected at the indicated time points post 4-OHT withdrawal. (d) Effect of miR-22 overexpression onTet1 expression in colony-forming cells with MLL-AF9, AE9a or FLT3-ITD/NPM1c+. (e) Expression ofTet1 in BM progenitor cells of 6-weeks old miR-22−/− or wild-type mice. (f) Effect of miR-22 overexpression on TET1 expression in THP-1 and KOCL-48 AML cells 48h post transfection. (g) Expression of pri-, pre- and mature miR-22 in BM progenitor cells of 6-weeks old Tet1−/− or wild-type mice. Mean±s.d. values are shown. *P<0.05, t-test.

http://www.nature.com/ncomms/2016/160426/ncomms11452/images_article/ncomms11452-f6.jpg

(a) Tet1 targets miR-22 promoter region (−1,100/+55bp), as detected by luciferase reporter assay 48h post transfection in HEK293T cells. (b) Expression of TET1/2/3, EZH2, SIN3A, GFI1 and miR-22 in THP-1 cells 72h post treatment with 1μM ATRA or DMSO control. (c) Co-immunoprecipitation assay showing the binding of endogenous GFI1 and TET1 in THP1 cells. (d) ChIP-qPCR analyses of the promoter region of miR-22 in THP-1 cells 72h post treatment with 1μM ATRA or DMSO. Upper panel: PCR site on the CpG-enriched region of miR-22 gene locus. Note: miR-22 is coded within the second exon of a long non-coding RNA (MIR22HG), which represents the primary transcript of miR-22. Lower panels: enrichment of MLL-N terminal (for both wild-type MLL and MLL-fusion proteins), MLL-C terminal (for wild-type MLL), TET1, EZH2, SIN3A, GFI1, H3K27me3, H3K4me3 or RNA pol II at miR-22 promoter region. (e) Expression levels of TET1, EZH2, SIN3A and miR-22 in GFI1 knockdown cells. (f) ChIP-qPCR analyses of the promoter region of miR-22 in THP-1 cells transduced with GFI1 shRNA or control shRNA. Enrichment of GFI1, TET1, EZH2 and SIN3A are shown. (g) Effects of knockdown of TET1, EZH2 and/orSIN3A on miR-22 expression. The expression level of miR-22 was detected in THP-1 cells 72h post transfection with siRNAs targeting TET1, EZH2 and/or SIN3A. Mean±s.d. values are shown. *P<0.05;**P<0.01 (t-test). (h) Schematic model of the regulatory pathway involving miR-22 in AML and ATRA treatment.

 

The miR-22-associated regulatory circuit in AML

         Restoration of miR-22 expression and function to treat AML

 

Figure 7: Therapeutic effect of miR-22-nanoparticles in treating AML.

http://www.nature.com/ncomms/2016/160426/ncomms11452/images_article/ncomms11452-f7.jpg

(a,b) Primary leukaemia BM cells bearing MLL-AF9 (a) or AE9a (b) were transplanted into sublethally irradiated secondary recipient mice. After the onset of secondary AML (usually 10 days post transplantation), the recipient mice were treated with PBS control, or 0.5mgkg−1 miR-22 or miR-22 mutant RNA oligos formulated with G7 PAMAM dendrimer nanoparticles, i.v., every other day, until the PBS-treated control group all died of leukaemia. (c) NSGS mice49 were transplanted with MV4;11/t(4;11) AML cells. Five days post transplantation, these mice started to be treated with PBS control, miR-22 or miR-22 mutant nanoparticles at the same dose as described above. Kaplan–Meier curves are shown; the drug administration period and frequency were indicated with yellow arrows. The P values were detected by log-rank test. (d) Wright–Giemsa stained PB and BM, and H&E stained spleen and liver of the MLL-AF9-secondary leukaemic mice treated with PBS control, miR-22 or miR-22 mutant nanoparticles.

We then tested the miR-22 nanoparticles in a xeno-transplantation model49. Similarly, the nanoparticles carrying miR-22 oligos, but not miR-22 mutant, significantly delayed AML progression induced by human MV4;11/t(4;11) cells (Fig. 7c). The miR-22-nanoparticle administration also resulted in less aggressive leukaemic pathological phenotypes in the recipient mice (Supplementary Fig. 6e). Thus, our studies demonstrated the therapeutic potential of using miR-22-based nanoparticles to treat AML.

 

It remains poorly understood how TET proteins mediate gene regulation in cancer. Here we show that in de novo AML, it is TET1, but not TET2 (a reported direct target of miR-22 in MDS and breast cancer15, 16), that inversely correlates with miR-22 in expression and negatively regulates miR-22 at the transcriptional level. Likely together with GFI1, TET1 recruits polycomb cofactors (for example, EZH2/SIN3A) to the miR-22 promoter, leading to a significant increase in H3K27me3 occupancy and decrease in RNA pol II occupancy at that region, and thereby resulting in miR-22 repression in AML cells; such a repression can be abrogated by ATRA treatment. Thus, our study uncovers a novel epigenetic regulation mechanism in leukaemia involving the cooperation between TET1/GFI1 and polycomb factors.

Besides GFI1, it was reported that LSD1 is also a binding partner of TET1 (ref. 50). Interestingly, LSD1 is known as a common binding partner shared by TET1 and GFI1, and mediates the effect of GFI1 on hematopoietic differentiation51, 52. Thus, it is possible that LSD1 might also participate in the transcriptional repression of miR-22 as a component of the GFI1/TET1 repression complex.

We previously reported that TET1 cooperates with MLL fusions in positively regulating their oncogenic co-targets in MLL-rearranged AML14. Here we show that TET1 can also function as a transcriptional repressor (of a miRNA) in cancer. The requirement of TET1-mediated regulation on expression of its positive (for example, HOXA/MEIS1/PBX3)14 or negative (for example, miR-22) downstream effectors in leukemogenesis likely explains the rareness of TET1 mutations in AML53, and highlights its potent oncogenic role in leukaemia.

The aberrant activation of both CREB and MYC signalling pathways has been shown in AML24, 25,26, 54, 55, but the underlying molecular mechanisms remain elusive. Our data suggest that the activation of these two signalling pathways in AML can be attributed, at least in part, to the repression of miR-22, which in turn, results in the de-repression of CRTC1 (CREB pathway), FLT3and MYCBP (MYC pathway), and leads to the upregulation of oncogenic downstream targets (for example, CDK6, HOXA7, BMI1, FASN and HMGA1) and downregulation of tumour-suppressor downstream targets (for example, RGS2).

In summary, we uncover a TET1/GFI1/EZH2/SIN3A⊣miR-22⊣CREB-MYC signalling circuit in de novo AML, in which miR-22 functions as a pivotal anti-tumour gate-keeper, distinct from its oncogenic role reported in MDS or MDS-derived AML16. Thus, our study together with the study of Song et al.16 highlight the complexity and functional importance of miR-22-associated gene regulation and signalling pathways in hematopoietic malignancies, and may provide novel insights into the genetic/epigenetic differences between de novo AML and MDS.

Our findings also highlight the possibility of using miR-22-based therapy to treat AML patients. Our proof-of-concept studies demonstrate that the nanoparticles carrying miR-22 oligos significantly inhibit AML progression and prolong survival of leukaemic mice in both BMT and xeno-transplantation models. Notably, miRNA-based nanoparticles have already entered clinical trials56. It would be important, in the future, to further test the combination of miR-22-carrying nanoparticles (or small-molecule compounds that can induce endogenous expression of miR-22) with standard chemotherapy agents (cytosine arabinoside and anthracycline), or with the emerging small molecule inhibitors against MYC and/or CREB pathway effectors, to achieve optimal anti-leukaemia effect with minimal side effects. Overall, our results suggest that restoration of miR-22 expression/function (for example, using miR-22-carrying nanoparticles or small-molecule compounds) holds great therapeutic potential to treat AML, especially those resistant to current therapies.

 

MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications  

Wed, July 13, 2016   The New York Academy of Sciences    Presented by the Biochemical Pharmacology Discussion Group
http://www.nyas.org/Events/Detail.aspx?cid=787a5d77-8354-4df7-92d5-91db18b2ce49

MicroRNAs (miRNAs) are single-stranded RNAs about 22 nucleotides in length that repress the expression of specific proteins by annealing to complementary sequences in the 3′ untranslated regions (UTRs) of target mRNAs. Apart from their posttranscriptional expression, or silencing, miRNAs may also direct mRNA destabilization and cleavage. Moreover, rather than targeting a single disease-associated protein target as many small molecule drugs and antibodies do, each miRNA may serve to repress the expression of numerous proteins involved in the pathogenesis and progression of various diseases and could therefore potentially interfere with multiple disease-promoting signal transduction pathways. Because aberrant expression of miRNAs has been implicated in numerous disease states, miRNA-based therapies have sparked much interest for the treatment of a variety of diseases. The objective of this symposium is to bring together investigators who have led the field in describing what miRNAs do and their potential in treating diseases, as well as those who are translating these findings into promising drug candidates, some of which have already advanced into early stage clinical trials.

Call for Poster Abstracts

Abstract submissions are invited for a poster session. For complete submission instructions, please send an email to miRNA@nyas.org with the words “Abstract Information” in the subject line. The deadline for abstract submission is May 13, 2016.

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Curator: Aviva Lev-Ari, PhD, RN

Sunitinib brings Adult acute lymphoblastic leukemia (ALL) to Remission – RNA Sequencing – FLT3 Receptor Blockade

Screen Shot 2021-07-19 at 7.36.02 PM

Word Cloud By Danielle Smolyar

http://pharmaceuticalintelligence.com/2012/07/09/sunitinib-brings-adult-all-to-remission-rna-sequencing/

 

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Curator: Aviva Lev-Ari, PhD, RN

Sunitinib brings Adult acute lymphoblastic leukemia (ALL) to Remission – RNA Sequencing – FLT3 Receptor Blockade

http://pharmaceuticalintelligence.com/2012/07/09/sunitinib-brings-adult-all-to-remission-rna-sequencing/

 

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Sunitinib brings Adult Acute Lymphoblastic Leukemia (ALL) to Remission – RNA Sequencing – FLT3 Receptor Blockade

Curator: Aviva Lev-Ari, PhD, RN

Sunitinib brings Adult Acute Lymphoblastic Leukemia (ALL) to Remission – RNA Sequencing – FLT3 Receptor Blockade

Word Cloud by Daniel Menzin

Updated 11/13/2013

Pazopanib versus Sunitinib in Renal Cancer

N Engl J Med 2013; 369:1968-1970November 14, 2013DOI: 10.1056/NEJMc1311795

Article

To the Editor:

Cancer treatments are expensive. The estimation of the total cost can be challenging because of several factors such as efficacy, toxicity, and the costs and duration of supportive care and end-of-life care. Motzer et al. (Aug. 22 issue)1 report similar efficacy but a favorable safety and quality-of-life profile and less medical resource utilization with pazopanib as compared with sunitinib in first-line therapy for metastatic renal cancer. Since oncology is becoming an increasingly value-based specialty, we wanted to highlight another important aspect of this trial. Pazopanib appears to be favorable not only in terms of safety and quality of life, but also in terms of overall cost. A 30-day supply of pazopanib (at a dose of 800 mg daily) ranges from $3,500 to $8,556, whereas a 30-day supply of sunitinib (at a dose of 50 mg daily) ranges from $4,500 to $13,559.2 The total cost of pazopanib during the median progression-free survival of 8.4 months is $29,400 to $71,870, and the total cost of sunitinib during the median progression-free survival of 9.5 months is $42,750 to $127,454. Less toxicity and less medical resource utilization with pazopanib will most likely further lower the overall costs of treatment with this agent. Comparative-effectiveness trials hold great promise for maximizing patient safety, improving treatment outcomes, and reducing costs.

Ryan Ramaekers, M.D.
Mark Tharnish, Pharm.D.
M. Sitki Copur, M.D.
Saint Francis Cancer Treatment Center, Grand Island, NE
mcopur@sfmc-gi.org

No potential conflict of interest relevant to this letter was reported.

2 References

To the Editor:

Motzer et al. report a combined analysis of two open-label noninferiority trials (927 patients in the original trial and 183 patients in a second trial), each of which compared pazopanib with sunitinib with respect to progression-free survival in renal-cell carcinoma. Quality-of-life outcomes were subjective.

Analysis of noninferiority trials is notoriously difficult.1,2 The authors’ analysis of the trials, which was open-label because of the different administration schedules of the drugs, presents problems in interpreting progression-free survival and quality of life. The studies define disease progression differently. The larger study defined progression-free survival according to independent review. The protocol for the smaller study states that progression-free survival “will be summarized . . . based on the investigator assessment.” Inference from subjective outcomes in unmasked trials (e.g., quality of life in both studies and progression-free survival in the smaller study and therefore in the combined analysis) is subject to well-known bias. Moreover, the article does not state how many of the 379 participants (34%) who discontinued the intervention before death or disease progression (see Fig. S2 in the Supplementary Appendix, available with the full text of the article at NEJM.org) were assessed for progression-free survival. A fair comparison must use rigorous methods to handle missing data.3 Since the article did not deal appropriately with missing data, its conclusions regarding noninferiority are uninterpretable.

Janet Wittes, Ph.D.
Statistics Collaborative, Washington, DC
janet@statcollab.com

Dr. Wittes reports that her company, Statistics Collaborative, has consulting agreements with both GlaxoSmithKline and Pfizer, the manufacturers of the drugs discussed in the article by Motzer et al. In addition, Statistics Collaborative has contracts with several other companies that produce drugs for patients with cancer. No other potential conflict of interest relevant to this letter was reported.

3 References

To the Editor:

Motzer et al. state that “the results of the progression-free survival analysis in the per-protocol population were consistent with the results of the primary analysis.” However, the predefined margin of noninferiority (<1.25) was not met. The upper limit of the confidence interval (1.255) was clearly above the defined threshold.1 In a noninferiority trial, the use of the intention-to-treat population is generally nonconservative,2 the full analysis set and the per-protocol analysis set are considered to have equal importance, and the use of the intention-to-treat population should lead to similar conclusions for a robust interpretation.3 Thus, it is surprising that the authors did not come to or discuss the same conclusions as that of the French National Authority for Health4: “serious doubt exists about the noninferiority result of pazopanib compared to sunitinib” and “the clinical significance of the noninferiority threshold defined in the protocol was an efficacy loss of 2.2 months in the median progression-free survival. This is too large for patients.”

Jochen Casper, M.D.
Silke Schumann-Binarsch, M.D.
Claus-Henning Köhne, M.D.
Klinikum Oldenburg, Oldenburg, Germany
casper.jochen@klinikum-oldenburg.de

Dr. Casper reports receiving consulting fees from Bayer, Novartis, and Pfizer and speaking fees from Novartis and Pfizer. No other potential conflict of interest relevant to this letter was reported.

4 References

The authors reply: In reply to Ramaekers et al.: we agree that decisions regarding the provision of health care include economic evaluations to identify treatments that provide the best clinical benefit at an acceptable cost.

To clarify a point in the letter by Wittes: the primary end point of this phase 3 trial was progression-free survival evaluated by an independent review committee; these data were assessed for all 1110 patients from both trials. This is specified in the protocol. The consistency of the quality-of-life results with the observed differences in the safety profiles for the two drugs speaks to the absence of bias in the quality-of-life outcome. The number of patients in whom follow-up ended before progression was assessed by the independent review committee was balanced between the two groups: 156 patients in the pazopanib group (28%) and 168 patients in the sunitinib group (30%). To Wittes’s final point regarding rigorous methods to handle missing data: the algorithm for assigning disease-progression and censoring dates followed the Guidance for Industry of the Food and Drug Administration1 and is included in the protocol of our article.

In reply to Casper et al.: there is no consensus regarding whether the per-protocol population is more conservative than the intention-to-treat population for the noninferiority analysis.2,3Reviews of noninferiority trials indicate that the per-protocol population is not generally more conservative than the intention-to-treat population, and there are scenarios in which the per-protocol analysis itself could introduce bias.3 A systematic review indicated that more than 70% of published findings from noninferiority trials in oncology show results in only the intention-to-treat population and not in the per-protocol population.4 Our phase 3 trial had a single primary analysis in the intention-to-treat population, with the per-protocol population included as a key sensitivity analysis, as supported by Fleming et al.5 No formal hypothesis testing was planned for the per-protocol population, nor was the trial powered for this. Consistency of the point estimates was desired to show an absence of bias due to the analysis population. This absence of bias was shown by the consistency of the hazard ratios (1.07 in the per-protocol analysis vs. 1.05 in the primary analysis). For an underpowered per-protocol comparison, it is inappropriate for Casper et al. to interpret that the upper bound that barely exceeded 1.25 in our per-protocol analysis is an indication of inconsistency of results across the two populations. The noninferiority margin was selected in consultation with oncology experts, and justification of the margin is in the protocol.

Robert J. Motzer, M.D.
Memorial Sloan-Kettering Cancer Center, New York, NY
motzerr@mskcc.org

Lauren McCann, Ph.D.
Keith Deen, M.S.
GlaxoSmithKline, Collegeville, PA

Since publication of their article, the authors report no further potential conflict of interest.

REFERENCES

Food and Drug Administration. Guidance for industry: clinical trial endpoints for the approval of cancer drugs and biologics. May 2007 (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm071590.pdf).
Jones B, Jarvis P, Lewis JA, Ebbutt AF. Trials to assess equivalence: the importance of rigorous methods. BMJ 1996;313:36-39[Erratum, BMJ 1996;313:550.]
CrossRef | Web of Science | Medline
Brittain E, Lin D. A comparison of intent-to-treat and per-protocol results in antibiotic non-inferiority trials. Stat Med 2005;24:1-10
CrossRef | Web of Science | Medline
Tanaka S, Kinjo Y, Kataoka Y, Yoshimura K, Termukai S. Statistical issues and recommendations for noninferiority trials in oncology: a systematic review. Clin Cancer Res 2012;18:1837-1847
CrossRef | Web of Science | Medline
Fleming TR, Odem-Davis K, Rothmann MD, Li Shen Y. Some essential considerations in the design and conduct of non-inferiority trials. Clin Trials2011;8:432-439
CrossRef | Web of Science | Medline
SOURCE

Original Article Published on 7/9/2012

July 6, 2012 NY Times reports on a new approach based on DNA and RNA sequencing and a cancer drug for kidney cancer to bring REMISSION to Adult acute lymphoblastic leukemia (ALL).

On the lower left corner of this page – Watch the VIDEO

second-chance.html

Dr. Lukas Wartman, is a Cancer Researcher specializing in Leukemia. He suspected he had Leukemia, the very disease he had devoted his medical career to studying.

After years of treatment and two relapses of ALL, he has exhaused all conventional approaches to his disease. At Washington University in St. Louis, his colleagues in the lab, decoded Dr. Wartman’s genetic information by genome sequencing techniques t determine the genetic cause of his ALL. The team found an overactive gne, FLT3 on Chromosome 13. The gene was treated with pfizer’s Suntinib drug for advanced kidney cancer.

Blood samples free of ALL found in days after using the drug. As results were very promising, Pfizer, the drug’s maker who has turned down Dr. Wartman’s request for the drug under their compassionate use program, though he explained that his entire salary was only enough to pay for 7 1/2 months of Sutent (Suntinib). While he does not know why Pfizer gave him the drug finally, he suspects it was the plea of his Nurse Practitioner, Stephanie Bauer, NP.

Identification of the genetic cause for his ALL, thus discovering a breakthough in understanding and treatment for ALL in other patients, involved the following steps:

SAMPLE

two tissue samples taken from Dr. Wartman’s Bone marrow and skin cells

SEQUENCE

Extracts of DNA and RNA from Dr. Wartman’s cells, two types of genetic material tested

COMPARISON

DNA sequesnces showed genetic mutations possibly related to his ALL, none seemed treatable. However, RNA sequencing revealed that a normal Gene, FLT3, on cheomozome 13, was overactive in his leukemia cells

TARGETING

The FLT3 gene helps create new white blod cells in the bone marrow. Dr. Wartman’s marrow bone cells were covered with an extreme number of FLT3 receptors which possibly caused the growth of his leukemia.

TREATMENT – Receptor Blockade 

Drug known to block FLT3 receptor, Sunitinib, used for kedney cancer treatment, was given to Dr. Wartman. Two weeks after Dr, Wartman began taking the drug, tests revealed that his leukenia was in remission.

NEW MARKETS FOR FLT3  GENE BLOCKADE DRUG  – KIDNEY CANCER AND LEUKEMIA

Pfizer has NOW a NEW market for Sunitinib — All CANCER PATIENTS DIAGNOSED WITH Adult acute lymphoblastic leukemia (ALL) where an overactive FLT3 gene on chomosome 13 is found.

NEW TREATMENT OPTIONS FOR Adult acute lymphoblastic leukemia (ALL)

Thus, any (ALL) diagnosed patient needs to be tested for Chromosome 13, ONLY rather then the entire genome sequencing of the Patient. If FLT3 is not found overactive, THEN proceed with entire genome sequencing of the Patient. IF another gene is overactive FIND DRUG FOR RECEPTOR BLOCKADE.

SIZING THE MARKET FOR FLT3 BLOCKADE DRUGS: KIDNEY CANCER vs LEUKEMIA

The Market for Adult ALL is much bigger than the market for kidney cancer. Thus, this discovery regarding the remission of Dr. Wartman’s remission following two relapses is so significant for Pfizer and for any patient with the diagnosis of Adult ALL.

I recommend the reader to click on the links and follow the reactions of the public to this article in The New York Times.

http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?pagewanted=all

Read HUNDREDS of Comments by Cancer Patients and the readers of The New York Times Health Section

http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?pagewanted=all#commentsContainer

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