High Content Screening Identifies Synthetic Lethality Of Retinoid Receptor Agonists In IKZF1-Mutated BCR-ABL1 positive Acute Lymphoblastic Leukemia

Expression of BCR-ABL1 is a hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). While CML is typically responsive to tyrosine kinase inhibitor monotherapy, BCR-ABL1-positive ALL is associated with a high frequency of treatment failure and relapse, even in the era of TKI therapy. Alterations of the lymphoid transcription factor gene IKZF1 (IKAROS) are frequent in BCR-ABL1 ALL and in CML at lymphoid blast crisis. Moreover, IKZF1 alterations are associated with poor outcome in BCR-ABL1-positive and -negative B-ALL. IKZF1 alterations are usually heterozygous and include loss-of-function deletions (∼60% of cases), focal deletion resulting in expression of a dominant negative isoform lacking the N-terminal DNA-binding zinc fingers, IK6 (∼30%) and missense mutations (∼10%). In addition, over half of BCR-ABL1-positive ALL cases harbor deletions of CDKN2A/B (INK4/ARF).

We used two murine models of BCR-ABL1 leukemia to examine the role of IKZF1 alterations (deletions and IK6) and ARF loss in disease lineage and treatment responsiveness. In the first model, lineage negative Arf^-/-, Ikzf1^+/- or wild type (WT) bone marrow was transduced with p185 BCR-ABL1-expressing retrovirus and either an IK6-expressing retrovirus or empty vector followed by transplantation into irradiated wild type mice. p185-transduced WT marrow invariably induced a CML-like leukemia, whereas Arf loss and Ikzf1 alterations synergistically drove an aggressive pre-B ALL. In a complementary model, unmanipulated Arf^-/-, Ikzf1^+/-, or WT marrow was transduced with p185 BCR-ABL and transplanted immediately or after in vitro derivation of pre-B cell cultures. In this model, Arf loss and Ikzf1 haploinsuffiency or expression of IK6 reduced the latency of ALL and reduced responsiveness to dasatinib in vitro and in vivo. Strikingly, perturbation of Ikzf1 activity by haploinsufficiency, expression of IK6, zinc finger missense mutations, or shRNA mediated knockdown was associated with increased expression of the stem cell marker CD90 (Thy1) and the growth of cells in large aggregates in liquid cultures. Ikzf1 alteration was also associated with induction of a hematopoietic stem cell-like gene expression program, and increased self-renewal manifested as enhanced serial colony forming potential. This aberrant growth and self-renewal phenotype was abrogated by neutralizing anti-Thy1 antibodies. Thy1 is a known IKZF1 target, and these findings suggest that IKZF1 alterations derepress Thy1 expression resulting in enhanced stemness and treatment resistance.

To identify agents that may enhance TKI responsiveness, we screened the activity of 384 FDA approved compounds in Arf^-/- BCR-ABL1+ IK6+ pre-B. We assessed cell viability using CellTiter Glo, and measured the number and size of cell aggregates by high throughput microscopy to identify drugs that selectively inhibit cell aggregation. This identified the retinoid X receptor agonist bexarotene as a potent inhibitor of cell aggregation. A follow-up screen of 128 nuclear hormone receptor effector compounds identified potent anti-aggregation activity of 9-cis retinoic acid, 13-cis retinoic acid, all-trans retinoic acid and carbacyclin. In contrast, N-oleoylethanolamine, an antagonist of perioxisome proliferator-activated receptors that heterodimerize with RXRs, markedly increased cell aggregation. All retinoid receptor agonists induced cell cycle arrest and reduced proliferation without inducing apoptosis, and attenuated Thy1 expression, abrogated colony formation and enhanced the efficacy of dasatinib in vitro and in mice transplanted with Arf^-/- p185 IK6 cells. Notably, Ikzf1is a predicted retinoid receptor target, and bexarotene treatment upregulated expression and nuclear localization of wild type IKZF1 in Arf^-/- p185 pre-B cells, with and without IK6.

Thus, IKZF1 alterations drive lymphoid lineage, and induce self-renewal and drug resistance in BCR-ABL1 ALL. IKZF1 alterations are associated with overexpression of Thy1, a target of IKZF1 transcriptional repression. This phenotype is reversed by retinoid receptor agonists that induce expression of IKZF1, attenuate Thy1 expression and increase sensitivity to TKI therapy. These data provide important new insights into the role of IKZF1 alterations in leukemogenesis, and identify a new therapeutic option in high risk B-ALL.