Targeting Survivin In Recalcitrant Acute Lymphoblastic Leukemia
Bibliographic record
Abstract
Abstract Abstract 3263 Background: Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), the development of drug resistance and long-term side effects of current treatments warrant new treatment modalities. Survivin/BIRC5, an inhibitor of apoptosis protein, is critical for the survival and proliferation of cancerous cells, is expressed in AML and ALL cells, and has been implicated in leukemia relapse. In the present study, we test the hypothesis that survivin is critical to the pathway of self-renewal of drug-resistant ALL cells. Methods: For gain of function studies, primary ALL cells were transduced with lentiviral survivin IRES GFP reporter (Survivin GFP) or empty GFP as a control. For loss of function studies, an inducible lentiviral shRNA vector expressing tRFP upon induction with doxycyclin was used. For in vitro evaluation of Survivin, CFU assays were used to monitor self-renewal capability, MTT assays and Trypan blue counts for viability determination were used for drug testing. For in vivo experiments, we used a NOD/SCID IL2Rγ−/- xenograft model with patient-derived ALL cells. Results: Survivin overexpression in primary ALL cells led in vitro to 4-fold more colonies than control in primary and secondary CFU assays and to increased resistance against Vincristine, Dexamethasone and L-Asparaginase (VDL) compared to controls (p<0.05). In vivo, animals injected with ALL cells overexpressing survivin died earlier of leukemia with a median survival time (MST) of 43 days (n=4) compared to control animals (MST=51.5 days) (n=4) (p<0.05). Therefore, overexpression of survivin increases self-renewal of patient-derived ALL cells in vitro and accelerates leukemia development in vivo. Conversely, in vitro inhibition of Survivin using shRNA decreased CFU compared to controls (p<0.0001). In vivo, when animals were injected with Survivin shRNA or non-silencing control shRNA and treated for 4 weeks with VDL, the combined VDL + Survivin shRNA treated group not only lived significantly longer (MST=213 days, n=3) compared to the control group (MST=117 days; n=3) (p<0.05) but remained disease-free until the end of follow-up (Day 213). Immunohistology and flow cytometry staining for huCD45+ cells in various organs showed the absence of leukemia cells in the VDL + shRNA treated group compared to the control, indicating that adjuvant knockdown of Survivin in combination with chemotherapy eradicates drug resistant primary leukemia. To further evaluate loss of function of Survivin in ALL, we used a survivin knockout mouse model. Survivinflox/flox bone marrow cells were retrovirally transformed with BCR-ABL1 p210 or MLL-ENL. Subsequent to leukemic outgrowth, cells were transduced with either GFP control or inducibly deleted using a Cre-GFP vector and GFP signal was quantitated by flow cytometry. Interestingly, Survivin deleted Cre-GFP positive oncogene transformed cells showed reduced proliferation compared to GFP controls (p<0.05), indicating that knockout of Survivin in murine leukemia is required for survival of leukemic cells. Finally, we determined the effect of pharmacological downregulation of Survivin using EZN-3042, a novel locked nucleic acid antisense oligonucleotide (LNA-AsODN) against Survivin. Single agent treatment of 6 primary ALL cases, with 20 mM of a scrambled LNA control (EZN-3088) or EZN-3042, were respectively assayed. Mean viability for EZN-3088 treatments was 78.6% ± 12.0% versus 44.1% ± 10.9% for EZN-3042 (p<0.001). EZN-3042 knockdown of Survivin was confirmed by Western blot. LNA treatment in combination with chemotherapy of a primary Philadelphia chromosome positive (Ph+) ALL resulted in a mean viability of 73.4% ± 1.8% for EZN-3088/Nilotinib versus 3.6% ± 0.5% for EZN-3042/Nilotinib (p<0.001). Primary Ph− ALL cells treated with EZN-3088/VDL resulted in a mean viability of 36.5% ± 0.5% versus 8.3% ± 4.3% for EZN-3042/Nilotinib (p<0.01). Conclusion: Taken together, we show that Survivin is a key component in primary drug resistant ALL cells and adjuvant specific targeting of Survivin using shRNA or EZN3042 has the potential to eradicate relapse of leukemia. Disclosures: Yang: Amgen Inc: Employment, Equity Ownership.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".