1. New insights into leukemic microenvironment in bone marrow Alexey Bersenev’s journal club Jan. 16, 2009

2. What is the leukemic niche? Why does it matter? Leukemic microenvironment in bone marrow Hypothesis 1: Similar to normal HSCs - LSCs support hierarchy and depend upon interactions within a specific niche, the LSC niche. Hypothesis 2: If any interactions are specific only for LSC and their niches, they could be a target for therapy What do we know about it?

3. Human AML LSCs (CD34+/CD38-) in NOG model: 1. home to the endosteal niche of the bone marrow ; 2. win “engraftment competition” with normal hematopoietic stem cells ; 3. rest in G0 cell cycle phase ; 4. m ore likely this mechanism explains why LSCs are chemotherapeutically resistant and cause AML relapse

4. 2. H uman mesenchymal stromal progenitor cells, derived from acute myelogenous leukemic bone marrow samples, have clonal genetic abnormalities (Michael Andreeeff, Blood (ASH Annual Meeting Abstracts) 2008 112: Abstract 2428 Right after winning the competition for the niche, cancer cells start to modify it, making it suitable for themselves. 1. Synergistic interactions between leukemic cells and osteoclast progenitors in bone microenvironment (Laura Calvi, Blood (ASH Annual Meeting Abstracts) 2008 112: Abstract 322) Abnormal cells in BM microenvironment and stroma:

5. Сell surface receptors expressed on HSC/progenitors, including CD44, ckit, CXCR4 and β1 integrins , seem to be vital for the localization and retention in the bone marrow Scheme of putative migration of stem cells between the bloodstream and bone marrow David A. Williams and Jose A. Cancelas (Nature 2006;444:828 )

6. 1. Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells. (Krause DS, et al, Nat Med. 2006 Oct;12(10):1175-80) 2. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. (Jin L. et al, Nat Med. 2006 Oct;12(10):1167-74) Targeting of CD44

7. Targeting of CXCR4 1. The CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. (Azab AK, et al. Blood online Jan. 12, 2009) 2. Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML (Michael Andreeff group, Blood (ASH Annual Meeting Abstracts) 2008 112: Abstract 2415

8. Questions: How do leukemic progenitors home and where do they stay in BM? How do normal HSC/progenitors interact with the leukemic microenviroment? How can we manipulate migration, homing and interaction of leukemic cells with their niches?

10. Few CD34+ cells co - localize with tumor upon homing to BM (day 0 ), yet CD34+ cells migrate into tumor niches over time

11. CD34+ cells in leukemic microenvironment do not response to G-CSF mobilization The malignant microenvironment, or niche, attracts CD34+ cells, but does not maintain CD34+ cell pool size or response to cytokine mobilization

12. CD34+ cell BM homing is inhibited by pertussis toxin (PTX) treatment in normal, but not leukemic mice Same results with AMD3100 CD34+ cells pre-treatment

13. AMD3100 does not restore CD34+ cell mobilization in leukemic mice.

14. SDF-1 ( red ) is highly expressed in the parasagittal sinusoidal region (CD34+ cell homing sites) of control mice Nalm-6–GFP cells ( green ) preferentially home to and proliferate in this area marked down-regulation of SDF-1 expression, here shown at 35 days after Nalm-6–GFP engraftment

15. CD34+ cells (white) home to the SDF-1–positive parasagittal vascular niches in control mice CD34+ cells aberrantly home to SDF-1+–negative, lateral regions in tumor ( green )–engrafted mice

16. In vivo imaging demonstrates increased SCF ( red ) expression in a tumor ( green ) - engrafted versus naïve mouse

17. SCF ( red ) expression is markedly up-regulated in ALL-engrafted versus control mice. CD34+ cells (white) localize to regions of high SCF expression

18. hSCF (brown) expression in tumor-engrafted BM Human BM biopsy Mouse model

19. Neutralizing treatment with anti-SCF inhibits CD34+ cell migration into tumor 7 days after CD34+ cell transplant into leukemic mice Same results in long-term (4 weeks)

20. Neutralizing anti-SCF restores G-CSF–mediated CD34+ cell mobilization in leukemic mice

23. "This is not a cure for leukemia , but it's one more tool. We like to hit cancer from all sides. This approach could potentially boost the immune system's response to the cancer by protecting the HPCs that are the source of mature immune cells. It could also maintain the patient's ability to tolerate treatment and to remain active." "If human stem cells respond in the same way as mouse cells do, it could buy us time to apply other therapies" D orothy S ipkins (U of Chicago) Citations: