July 5th 2008 post. For an introduction to IRF4 please see my June 27^th post. Now, let’s see, the full “Nature” study starts with a discussion of a genetic method to identify therapeutic targets in cancer in which small hairpin RNAs (shRNAs) that mediate RNA interference are screened for their ability to block cancer cell proliferation and/or survival. The researchers used myeloma cells lines from three molecular subtypes.

 

Hairpin whaaats? Molecular subwhaaaats? I had to look up all this stuff even though I suspected that the latter had to do with all the different types of myeloma (truth be told, I didn’t find out that there were different types of myeloma until quite recently…in fact, before my diagnosis, I used to think that leukaemia was just one type of cancer…who’d ever heard of Hairy Cell Leukemia, Acute Myelogenous Leukemia or Acute Megakaryocytic Leukemia? Indeed, come to think of it, who’d ever heard of…Multiple Myeloma??? Ahhh, how things change…). Anyway, I found a lot of information about different myeloma subtypes online. I am not going to bother with small RNA hairpins…I never use hairpins anyway…well, okay, I used ‘em on my wedding day, nine years ago…but that is IT!

 

Myeloma subtypes. A UAMS communication (http://tinyurl.com/ssheh) tells us that in 2006 seven genetic subtypes of myeloma were identified among 414 myeloma patients. Of those seven subtypes, four were associated with better patient outcomes following high-dose chemotherapy and a stem cell transplantation. Ok, but what happened to the other three? I am curious. At some point I really should take a look at the full study, which is available for free online: http://tinyurl.com/3eqm6l

 

I also read the presentation (see: http://tinyurl.com/3efgjp) given by Dr. Rafael Fonseca at the island of Kos 2007 International Myeloma Workshop. Myeloma, he says, is not a homogenous type of cancer, but many well defined variants exist. And these variants can best be examined by looking at genetic/cytogenetic markers. The idea is that genetic testing should be useful in figuring out what kind of treatment would be the best for a particular subtype. Targeted treatment, in other words. Interesting concept…that we will come across again later…

 

Back to the IRF4 study. It is very technical, and I confess that I got lost at times among the various translocations and missense substitutions and fourth introns and coding regions…but a few things were clear, such as the following: The knockdown of IRF4 killed ten myeloma cell lines, but had a minimal effect on five lymphoma cell lines. Knockdown, by the way, is simply a technique used to reduce the expression of one of more genes. For instance, remember the mutant tumour-suppressor p53? Well, when this evil mutant form is “knocked down,” cancer cells become less aggressive. Anyway, the upshot is that ten myeloma cell lines were annihilated when IRF4 was knocked down. Excellent!

 

The researchers point out that the myeloma cell lines they tested bear many of the recurrent genetic aberrations typical of this cancer, including genetic abnormalities that activate the NF-kB pathway. Ah. So here we have another thing that triggers the infamous NF-kB…good to know, I suppose (sigh). Reading this study made me wish to have more genetic testing…this may be another matter I will discuss with my haematologist this fall (as much as I hate BMBs, it might be worth having another one…).

 

Back to the study. The myeloma lines tested were like drug addicts badly in need of a fix, which was provided by a perfectly normal (not aberrant, that is) IRF4. The researchers wanted to understand the molecular basis for this dependence, so they looked at genetic changes in the myeloma lines after IRF4 was knocked down. They noticed that 308 genes became down-regulated (down-regulation is the process whereby a response to a stimulus is reduced or suppressed. For instance, curcumin down-regulates the hyperactive transcription factor NF-kappaB in cancer cells).

 

The researchers then examined normal haematopoietic (= blood or blood-forming) cells that require high IRF4 expression, including plasma cells […] and dendritic cells, and found that IRF4 regulates a broader set of genes in myeloma than in individual haematopoietic subsets. So myeloma gives IRF4 the chance to expand its standard genetic network. And Mr. Normal Nice Guy IRF4 thus becomes Mr. Normal But Not So Nice Guy IRF4.

 

The paragraph continues: Roughly one-quarter of the IRF4 target genes in myeloma were upregulated in activated B cells but not plasma cells, including genes known to be important in cellular growth and proliferation, such as MYC. I looked up MYC on Wikipedia and discovered that the mutated or over-expressed form of this gene can cause cancer. 

 

I also found the following: Myc is activated upon various mitogenic signals such as Wnt, Shh […]. Activated by Wnt and Shh…wait a sec, those are signaling pathways that are crucial for the survival of cancer stem cells! Could cancer stem cells somehow be involved, here? There is no mention of cancer stem cells in the study so I have no way of answering that question. The more I think about it, the more it seems doubtful…oh well.

 

Anyway, according to the IRF4 study, MYC has a prominent role in the pathogenesis of myeloma. But when the activity of IRF4 was reduced, the levels of MYC mRNA also decreased by more than twofold in myeloma cell lines and caused MYC DNA-binding activity to decrease in nuclear extracts of myeloma cells. So it would seem that if you block IRF4 you also block MYC (and vice versa), and this process will eventually lead to the death of myeloma cells.

 

The researchers also discovered that IRF4 and MYC are co-dependent: Our data suggest that the oncogenic activation of MYC in myeloma upregulates IRF4, which in turn drives expression of MYC and other IRF4 target genes. A vicious cycle, in other words. Probably not an easy cycle to interrupt, I would imagine.

 

Another interesting sentence: […] the dependency of myeloma on IRF4 may be best described as ‘non-oncogene addiction’; that is, the aberrant function of a normal cellular protein that is required for cancer cell proliferation or survival. The loss of IRF4, they add, results in ‘death by a thousand cuts’. I like the sound of those last five words!

  

Another important observation: the reduction of the activity of IRF4 by only 50% (!!!) is enough to kill myeloma cells without harming healthy cells: a ~50% knockdown of IRF4 mRNA and protein was sufficient to kill myeloma cell lines. Wow.

 

The researchers are optimistic. At the end of the study they speculate that an IRF4-directed therapy might kill myeloma cells while sparing normal cells, and hope that IRF4 can be exploited as an Achilles’ heel of multiple myeloma. Too early to tell…but interesting.