Inactivation of the Arf Tumor Suppressor in Mouse BCR-ABL(+) B Cells Greatly Increases the Frequency of Leukemia-Initiating Cells and Confers Imatinib Resistance In Vivo.

Deletion of the intimately linked INK4A and ARF tumor suppressor genes (CDKN2A) frequently occurs in BCR-ABL-induced [Ph+] acute lymphoblastic leukemia (ALL) and correlates with their poor therapeutic response. Although BCR-ABL cancels the dependence of cultured primary murine pre-B cells on interleukin-7 (IL-7), it efficiently induces an Arf- and p53-dependent checkpoint that limits further cell expansion. In contrast, immortal Arf-null pre-B cells resist BCR-ABL-induced apoptosis. Thus, the combination of BCR-ABL expression and Arf inactivation confers synergistic properties of cytokine independence, resistance to apoptosis, and limitless replicative potential. To determine the impact of Arf inactivation on ALL development, we infected unconditioned bone marrow cells from Arf^+/+ or Arf-null mice with retroviral GFP-encoding vectors co-expressing either the p185^BCR-ABL or p210^BCR-ABL isoforms (hereafter p210 and p185). After short-term culture (8 days) under conditions that select for the outgrowth of pre-B cells, BCR-ABL(+) cells of both Arf genotypes were infused intravenously into immunocompetent syngeneic mice. Only Arf-null donor cells induced lympholeukemias. Remarkably, while 2 ×10⁵Arf^+/+ p210+ or p185+ pre-B cells failed to establish leukemias in recipient mice, as few as 20 Arf-null p185+ cells generated lethal lympholeukemias within 30 days. Signs of highly aggressive disease included splenomegaly, significant replacement (60–90%) of bone marrow with GFP(+) cells, and the emergence of numerous circulating GFP(+) lymphoblasts (up to 10⁵ per ul). Leukemic cells recovered from the bone marrow of moribund animals could be continuously cultured in vitro and maintained their pre-B cell immunophenotype and leukemogenic potential when retransplanted. Thus, in comparison to BCR-ABL(+), Arf^+/+ donor pre-B cells, their Arf-null counterparts are at least 10⁴-fold enriched in leukemia-initiating cell activity. Cultured BCR-ABL(+), Arf-null pre-B cells are exquisitely sensitive to the cytostatic effects of the BCR-ABL kinase inhibitor, imatinib. However, mice inoculated with as few as 200 BCR-ABL(+), Arf-null donor cells failed oral high-dose imatinib therapy (100 mg/kg bid). Equally surprisingly, tumor cells recovered from treated, moribund mice retained the same sensitivity to the drug (IC₅₀ < 150 nM) as the original donor cells, indicating that imatinib resistance in this setting is tumor cell-extrinsic and does not depend upon mutations or amplification of the BCR-ABL kinase. Imatinib restores the requirement of BCR-ABL(+) cells for IL-7, and conversely, exposure to saturating levels of IL-7 can significantly reduce imatinib sensitivity. Therefore, host-derived factors, such as IL-7, within hematopoietic tissues may rescue Arf-null leukemic cells from imatinib-mediated growth inhibition. Selective inhibition of Janus (JAK) kinases that transduce signals from cytokines like IL-7 can restore imatinib sensitivity in vitro, suggesting that targeted inhibition of cytokine signaling might play an adjunctive role in therapy of imatinib-refractory BCR-ABL-induced ALL.