Friday, April 07, 2006

micro RNAs

Now this is not for the faint-hearted. If it's too technical read the one about Irish politics instead.

In my lab we have been working on 13q14 longer than anybody else. My colleague David Oscier gets the credit for this. He published a paper detailing the first report of deletions at this site in hematological malignancies. It is not only important in CLL, but it also the most important chromosomal abnormality in multiple myeloma as well. He also found the deletion in some cases of myelofibrosis. (Fitchett et al Cancer Genet Cytogenet. 1987 24:143-50).

The chromosome lab that he set up was the first to regularly get good results with conventional karyotyping in CLL so that the original paper demonstrating that del 13q was a good prognostic feature in CLL and that there was a hierarchy of prognoses depending on the karyotype was largely composed of his patients (Juliusson et al N Engl J Med. 1990 323:720-4).

He isolated the bit of chromosome 13 that was always missing and sequenced the length of DNA that was missing. He also identified the genes that are represented on this missing piece of DNA. (Hawthorn et al Oncogene. 1993 8:1415-9. Chapman et al. Oncogene. 1994 9:1289-93. Liu et al Oncogene. 1997 15:2463-73) .Now here's the rub. Everybody expected the mechanism to be that of the classic tumor suppressor gene.

Retinoblastoma (which is also located at 13q14) is the classical example. This is a tumor of the eye in children that is caused by the loss of the Rb1 gene from both chromosome 13s. Usually the child inherits one mssing or damaged Rb1 gene from one parent and loses the other in childhood. Loss of both tumor suppressor genes allows the tumor to grow, but while there is one still intact, there is no tumor.

It was expected that there would be another gene at 13q14 that acted in a similar way to cause CLL. Although some CLLs do indeed have a deletion on both chromosome 13s, this is not the common finding. Never mind, we thought, although the other gene is there, when we sequence the area we will find a crippling mutation on the other chromosome. There are not many genes in this area. Depending on how you count them there are no more than 5, and possibly as few as two, so it was not going to be difficult to find a mutation. To cut a long story short there were no mutations, nor were there any epigenetic mechanisms like aberrant methylation that would account for both chromosomes not working (Corcoran et al Blood. 1998 91:1382-90. Kapanadze et al FEBS Lett. 1998 426:266-70. Grand et al Genes Chromosomes Cancer. 2004 40:78-83).

I guess people began to think that a small lab like ours had made a mistake, but the really big labs like Carlo Croce's and Ricardo Dalla-favera's confirmed David's findings.

Then Croce reported on microRNAs (miR). miRs are small lengths of single stranded RNA that are able to bind to messenger RNA to prevent its being translated into protein. They are part of the normal control system which allows cells to express or not express particular genes. In CLL Croce found that two miRs, miR-15a and miR-16-1, which are normally located at chromosome 13q14.3, are down-regulated in about 65% of patients (Cimmino et al PNAS 2005 102:13944-9).

The messenger RNA that these miRs target is the one that codes for bcl-2. They normally help to switch off bcl-2 production.

Bcl-2 is one of the complex series of proteins that controls apoptosis or programmed cell death. It happens to be one of the most important inhibitors of apoptosis. It is very important in the functioning of B cells. During an immune response against a foreign germ it is necessary to make a lot of antibody, and the right antibody at that. A process of rapid somatic mutation takes place in lymph nodes and spleen, as genes are shuffled and mixed, altered and corrected so that the appropriate antibody is made. This is a random process and a lot of nonsensical or even harmful antibodies are made. It is very important that the cells makeing these antibodies are killed. They don't need any bcl-2 around preventing that; hence the miRs to switch it off. However, the good antibody producing cells need to survive, and in them bcl-2 needs to be upregulated - but only for a while; when the germs have been dealt with we need the cells used in the immune response to die back, so the bcl-2 has to be switched off. Transgenic mice have been made in which the bcl-2 gene is always switched on. These mice grow huge lymph nodes and spleens - not because they have tumors, but because their lymphocytes are immortal and they bkeep accumulating.

Of course in CLL the lack of these specific miRs to switch off bcl-2 means that the CLL cells don't die properly either.

I am writing about miRs because of a paper that appeared recently in Cell. This concerns miR223 (yes there are a lot of them). Acute promyelocytic leukemia (APL) is quite a rare leukemia (sometimes called M3) where the leukemic blasts begin to mature but get stuck at the promyelocyte level. It is important because most patients get disseminated intravascular coagulation, and used to bleed to death. These patients almost always have a chromosomal transocation between chromosome 15 and 17 (t15;17) which puts the APL gene next to the RAR gene. It was a Chinese discovery that these patients respond well to a form of vitamin A known as all trans retinoic acid (ATRA). Treatment with ATRA has revolutionized the outlook for APL patients, and now it is one of the best types of acute leukemia to have, if you have to have leukemia.

In the Cell paper (2005; 123:819-31), Farzi and colleagues from Rome describe a series of experiments that help to sort out how ATRA works and what the function of vitamin A is in the normal development ofwhite cells. There are two transcription factors that compete to bind the promoter of miR223, NFI-A and C/EBPalpha. These two factors have long been known to regulate the growth and differentiation of many cell types.

This is how they interact. NFI-A keeps miR223 at low levels, but when ATRA is added the NFI-A is replaced by C/EBPalpha. miR223 the suppresses NFI-A. In APL, introduction of the gene for miR223 so that the miR is expressed cause the cells to overcome the block in diferentiation, while knocking out the miR223 blocks their ability to differentiate in response to ATRA. Some cases of APL become resistant to ATRA, and an understanding of this mechanism offers a way forward.

These microRNAs have allowed us a greater understanding of the regulation of genes and it would not be difficult to think of how small molecules could be adapted as drugs for more precise management of leukemias.


Anonymous said...

I love these types of posts. The functioning of the immune system is very complex (not just my opinion), and much remains to be uncovered.

I am puzzled by one thing. The Chiorazzi heavy hydrogen paper showed that CLL is a balance between proliferation and apoptosis, and that apoptotic rates seem to be normal in CLL patients; instead, it is the increase in proliferation that leads to bad outcomes.

Is it proliferation or defective apoptosis that is the cause of progressing CLL?

Terry Hamblin said...

All CLL cells have defective apoptosis, but by itself this only leads to a slow accumulation. There has to be a proliferative problem also for there to be a clinical problem with CLL.

Anonymous said...


I am curious about the ATRA in this post. A Dr. Gopal (fred Hutch-Seattle)is doing a clinical trial with Rituxan+Fenretinide (sp?). It is my understanding that the fenretinide is a vita A type of drug? Anthing close to your ATRA? Thanks as always for your time, focus and care.