SLIDES & TRANSCRIPTS
Monday, May 12, 2003

Apoptosis MDR in ALL: Significance and Exploitation

Michael Andreeff, M.D., Ph.D.

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TOP

I will briefly review apoptosis and multi-drug resistance. Here is the mitochondria. As you can see, which releases cytochrome C which activates caspase-9 and caspase-3, and this leads to apoptosis.

This is triggered by chemotherapy via p53 and bax, inhibition by BCL2, BCL-XL, and other anti-apoptotic proteins.
Interestingly, caspase-2 is now upstream of mitochondria, not downstream, and this has some implications.

Now, other factors than cytochrome 3 are released, in particular AIF and, in the nucleus G, they can induce DNA fragmentation without going through caspases, and this might be important, too, for drug development, because the caspase pathways are blocked.

So, this was the internal pathway in 30 seconds. The extrinsic pathway is TNF receptor of Fas-mediated, activates caspase-8, which activates caspase-3. This pathway is inhibited by IAPs, inhibitor of apoptosis proteins and, in particular, XIP and survivin.

It is also inhibited by Smac/DIABLO, which is released by the mitochondria. This pathway is linked to the mitochondria pathway through BID and BAK. So, there is cross talk between these two pathways.

Another inhibitor is COP, recently identified has not been examined in leukemia so far. These pathways intersect with signaling.

Here is the ras raf pathway, and they all give us targets and, of course, PI3 kinase AKT.

One of the interactions is the phosphorylation of BAD, as shown here, and the upregulation of survivin and XRAP by AKT signaling or MAP kinase signaling.

This gives us particular opportunities to interfere with cell survival.

TOP

So, reviewing the literature on the prognostic impact of BCL-2 and BAX, here are lousy data. It is not 100 percent convincing. Some authors contradict each other.

There is some evidence that increased BCL-2 is related to drug resistance in ALL. The largest series by Uckun showed no correlation between BCL-2 levels and achieving the formation of N3 survival, and Campana has similar data.

However, a paper from the German group found that BAX /BCL2 ratios were decreased at relapse.

TOP

I show their data here briefly.
These are paired samples, matched samples from the same patient diagnosis and relapse, a small series,

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but then confirmed in a large unmatched series.
So, BAX drastically is decreased and relapse BCL2 is not changed, so the BAX BCL2 ratio goes down, and this was confirmed in over 90 patients here at diagnosis and relapse.

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Interestingly, when we looked at spontaneous activation of caspase 3, there was loss of caspase 3 activation in the relapse cases. Obviously, that pathway is blocked in relapse, and again, this would provide therapeutic opportunities.

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This is supported by data from Cleary who showed that transformation of BCL progenitors by E2A-HLF requires a core expression of BCL2. Otherwise, one does not get leukemia.

An interesting observation is also the presence of BAX mutations that result in loss of immune detectable protein, and that could explain some of the data shown previously.

TOP

However, another study showed a very low incidence of BAX mutations in primary samples, not in cell lines.
The next pathway, intrinsic pathway here, trail, fas ligand, caspase 8, caspase 3.

TOP

Fas is very lowly expressed, if at all, in most primary leukemias. This includes AMLs, and certainly in ALLs by RT-PCR.

We round no detectable fas but, by nested RT, he found some splice variants. This confirms this data, and then the issue of fas mutations came up, but they found only two out of 81 T-ALLs with fas mutations. So, it is probably not a major contributor here to resistance of this pathway.

Interestingly, the Philadelphia chromosome positive ALL is very sensitive to TRAIL, while M.D. Anderson showed that PB ALL is completely insensitive to TRAIL and that is also true for AMLs, by the way.

TOP

So, there is a connection between the two pathways I mentioned initially. Fas signaling activates caspase-8 which activates BID and activates BAK, and that then feeds into the intrinsic pathways, and it is important to understand that these two pathways are not completely separate.

TOP

All the studies that we are doing in leukemias and ALL are usually done in suspension cultures, and it is really important to switch this.

Campana has done this some time ago, and cytokines alone cannot support survival of ALL cells. It is the contact with stroma cells and STF1 is the critical factor here, which results in the up-regulation of BCL-2 in the ALL, and we found the same in AMLs.

So, stroma cells protect B cell ALL from chemotherapy and I suggest in the future, when we test ALLs for chemosensitivity, we do this in co-cultivation on stroma cells, and not in suspension cultures.

TOP

Very briefly, A,B,C transporters, MDR is detected and expressed in ALLs. LRP and MRP are present in low levels. Their prognostic importance is very uncertain. MDR was found to be unfavorable in T cell ALL.

On the bottom you find probably the best paper on 203 patients by FTafuri, an Italian group, where MDR was expressed in 22 percent of cases, and patients who had MDR only had a SCR rate of 54 percent versus 80 percent in MDR negative cases. So, MDR may be of some importance.

TOP

This gives us targets, BCL-2, antisense, oligonucleotides and small molecule inhibitors. siRNA, I think, is nice, but not on the horizon yet.

TOP

This is the first small molecule BCL-2 inhibitor, HA14,

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which induces apoptosis in primary lymphoid leukemic cells, as shown here.

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It interferes with BAX heterodimerization, as shown here by IP, this BAX system, but the BCL2, there is a decrease in heterodimerization using the small molecule.

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There are new compounds now that are active in the sub-micromolar level, between 100 and 500 nanomoles and induce apoptosis by themselves without any addition of chemotherapy so far.

TOP

Fas TRAIL as a target is very questionable. I do not think that trail trials have still not started probably because of liver toxicity. Fas is very liver toxic, so I don't see a real possibility there, but I have been wrong before.

Now, caspase as a target, I mentioned survivin and XIP.

TOP

So, survivin inhibits caspase-3 and -7. XIP inhibits caspase-3, -7 and -9,

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and by just downregulating XIP by using specific antisense, one can induce apoptosis and decrease cell numbers.

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The alternative approach would be to use small molecule inhibitors that fit into the binding pocket between XIP and caspase ERBA2 domain as shown here.

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John Wheat's lab, Allen Schimmer has developed small inhibitors that are very active and induces apoptosis here in Burkitt cells, compound 34.

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Alternatively, one can also down-regulate survivin, again here shown by antisense, again by itself, and uses apoptosis. So, the inhibition of an inhibitor is sufficient to induce apoptosis in leukemic cells.

TOP

We found that inhibition of both BCL2 and MAP kinase was extremely potent in inducing apoptosis. So, MAP kinase inhibition, which is downstream of ras signalling, induces major apoptosis in the myeloid leukemias, with no effect on normal progenitor cells.

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When you combine this with BCL2 inhibition, either by antisense or small molecule inhibitor, we get truly synergistic effector combination indices of .3 to .1, something we have never seen by chemotherapy. This is all without any chemotherapy.

TOP

The next target would be interfering with the stroma, leukemia cell interaction. HDAC inhibitors that repress CXCR4 which is required for homing of stem cells to their stroma.

Another interesting observation was that BCR-ABL positive ALL were very sensitive to farnesyl transferase inhibition, of course. One would expect that Gleevec is important here, and clinical trials for that are underway.

MAP kinase can be inhibited by CR1040, and PI3 kinase inhibitors are under development.

TOP

MDR, of course, has been tested sufficiently in myeloid leukemias, not so much in lymphoid leukemias, as far as I know.

TOP

My last target is PPR gamma. PPR gamma is a nuclear transcription factor, over-expressed in all leukemias and not in normal stem cells. PPR gamma heterodimerizes with RXR.

So, the biggest advance in AML therapy, of course, was ATRA, which targets RAR, and we are trying to repeat this here in ALLs.

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The activation of these receptors with specific ligands results in signaling of apoptosis and differentiation.

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We have a compound called CDDO, a triterpenoid. This is a patient that is chemoresistant to Burkitt's lymphoma leukemia, who was super sensitive, as you can see, to one and two micromolar of CCDO concentration we can easily achieve in mice, at least.

So, we think that targeting this particular pathway is of potential benefit, and the possibility to, at the same time, activate RXR with Targretin is very intriguing. We are starting a clinical trial in leukemias, actually, this week at M.D. Anderson.

So, these are my targets based on apoptotic and MDR resistance. Thank you.

TOP