Chronic lymphocytic leukemia (CLL) represents the most prevalent adult leukemia in western countries with an accumulation of malignant, mature CD5+CD19+ B-lymphocytes in the blood, bone marrow (BM), and secondary lymphoid organs. Although standard frontline therapy consists of a cocktail of monoclonal antibody therapies directed against CD20 or CD52 surface antigens, and chemotherapy, these modalities are not curative. Given the necessity of B-cell receptor (BCR) signaling in CLL cell survival, small molecule inhibitors targeting BCR pathways have shown promising outcomes in clinical studies. These successes include recent FDA approvals of the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, and the phosphoinositide 3-kinase (PI3K) δ inhibitor, idelalisib. Despite the promise of these newly approved agents, refractory/relapsed CLL remains a challenge. While most patients initially respond to treatment, relapse is usually inevitable. The mechanism of relapse is still elusive – partly attributed to both an ineffective targeting of the mature leukemic burden and potential “leukemia initiating precursors” that may persist in CLL patient bone marrow.
This dissertation research is presented in two parts. The first part describes studies evaluating the in-vitro and in-vivo therapeutic efficacy of micro RNA 29b (miR-29b), a factor that is associated with under-expression in highly aggressive CLL, using a novel targeted delivery formulation in a novel mouse model of CLL. The second part evaluates the potential oncogenic properties of CD34+CD38-Lin- hematopoietic stem cell (HSC) from CLL patient bone marrow that gives rise to CD5+CD19+ “CLL-like disease” in immunocompromised NOD-scid IL2rγnull (NSG) mice. We also use a novel 3D micro-electroporation platform (MEP) to identify a rare stem cell sub-population in CLL patient bone marrow HSCs using molecular beacons and evaluate relevant therapies in vitro. The summary of these studies is outlined below.
The oncofetal antigen ROR1 (receptor tyrosine kinase orphan receptor 1) is expressed on malignant CLL cells, but not normal B cells, and serves as a unique target for CLL. Low expression of miR-29b observed in aggressive CLL is associated with poor prognosis and suggests that miR-29b can be used as a potential therapeutic agent. To facilitate selective and efficient delivery of miR-29b to CLL cells, we developed an ROR1 based immuno-nanoparticle delivery formulation for miR-29b selectively targeted to B-CLL but not normal B cells. The targeted delivery of miR-29b led to downregulation of DNMT1 and DNMT3A, modulated global DNA methylation changes, decreased SP1 and increased p21cip1 expression in ROR1+ cell lines and primary CLL cells in vitro. Furthermore, using an Eμ-TCL-1 mouse model expressing human ROR1, we demonstrated that miR-29B delivery enhanced overall survival by mediating cellular reprogramming via the downregulation of DNMT1 and DNMT3A. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21cip1 expression following targeted miR-29b treatment. This was confirmed at the protein level leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in a p21cip1-dependent compensation of the miR-29b effect on cell cycle arrest. These data form a basis for leukemia-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.
Acquired mutations have been reported in CLL bone marrow (BM) hematopoietic stem cells. This prompted us to attempt to trace the origin of oncogenesis during CLL hematopoiesis. In patients, disease-associated mutations observed in mature B-CLL are relatively rare in HSC and progressively accumulate throughout subsequent B-lymphoid developmental stages. Interestingly these mutations are restricted to B-lineage cells and are not detected within the myeloid lineage. Engrafting HSCs from CLL patients into immunocompromised NSG mice initiated a CLL-like disease phenotype with CD5+CD19+ B lymphocytes, although these were distinct from the original CLL clones as evidenced by VDJ usage, cytogenetic, and mutational status. These findings suggest that oncogenic lesions could originate in a subset of HSCs in the CLL BM, while the accumulation of additional mutations during early B-cell differentiation leads to disease progression.
Currently, there is no specific marker that can distinguish leukemogenic HSCs from normal HSCs in CLL. In this study, we tried to identify and target putative “CLL leukemia initiating cells”. Comparing to normal donors, we observed higher GATA2 expression in the HSCs from CLL-BM, which correlated with high levels of IKAROS family zinc finger 1 (IKZF1). Lenalidomide, an immune modulatory agent that has been shown to effectively downregulate IKZF1 in mature CLL, has a potential utility in targeting IKZF expressing putative “CLL leukemia initiating HSCs”.
To identify and evaluate the drug sensitivity of rare HSC subpopulations in CLL bone marrow, herein, we designed an array-based 3D microchannel electroporation platform (MEP) to deliver molecular beacons (MBs) into individual live cells, allowing the characterization of rare populations based on intracellular RNA detection. Using this device, CD34+CD38-Lin- HSCs from CLL patients— identified by intracellular GATA2 expression using the MEP platform with GATA2 MB— were evaluated for sensitivity to clinically relevant chemotherapeutic agents such as fludarabine, lenalidomide, and OSU-2S, a novel phosphatase activator we have identified to be effective in CLL. We found that GATA2+ HSCs from CLL-BM showed the higher drug resistance than GATA2- cells at 24 hr. The role of GATA2 in fludarabine resistance in these cells was further confirmed by siRNA-mediated knockdown of GATA2 in the GATA2+ cells. GATA2+ HSCs exhibited higher sensitivity to lenalidomide mediated degradation of IKZF1 compared to GATA2- cells. Our data suggest the heterogeneous GATA2+ HSC subpopulation in CLL-BM is poised for fludarabine resistance but may potentially by more sensitive to and be targeted by lenalidomide.
Our work, for the first time, provides a novel approach for targeting ROR1+ B-cell malignancies by selectively reprogramming the epigenetic framework of CLL cells in vivo. Additionally, we carried out a systemic investigation to identify a rare HSC subpopulation in CLL-BM that revealed a differential response to therapeutic agents in vitro and leukemogenic ability in vivo.