Yi Shan

A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia

A large-scale RNA interference screen reveals a new mechanism of imatinib resistance in chronic myeloid leukemia that can be therapeutically targeted. An Irresistible Combination Unlike most cancers, which have variable mutation patterns, chronic myeloid leukemia is normally associated with a specific genetic alteration, which produces a fusion protein called BCR-ABL. Famously, this protein was targeted with the first cancer-specific drug, known as imatinib (Gleevec), which is still the standard therapy for this cancer. Unfortunately, leukemia cells can develop resistance to imatinib, which does not always require mutations in BCR-ABL. Now, Ma et al. have identified a mechanism for imatinib resistance in chronic myeloid leukemia cells that lack mutations in BCR-ABL. The authors also demonstrated that a U.S. Food and Drug Administration–approved drug called trametinib can overcome this resistance and kill leukemia stem cells without harming the nonmalignant precursors that give rise to normal blood cells. Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL–independent IM resistance remains to be elucidated. To gain insight into BCR-ABL–independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL–independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration–approved MEK inhibitor, synergistically kills BCR-ABL+ IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL–independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL–independent IM resistance in CML and CML stem cells.

A tumor suppressor function of the Msr1 gene in leukemia stem cells of chronic myeloid leukemia

We have shown that Alox5 is a critical regulator of leukemia stem cells (LSCs) in a BCR-ABL-induced chronic myeloid leukemia (CML) mouse model, and we hypothesize that the Alox5 pathway represents a major molecular network that regulates LSC function. Therefore, we sought to dissect this pathway by comparing the gene expression profiles of wild type and Alox5(-/-) LSCs. DNA microarray analysis revealed a small group of candidate genes that exhibited changes in the levels of transcription in the absence of Alox5 expression. In particular, we noted that the expression of the Msr1 gene was upregulated in Alox5(-/-) LSCs, suggesting that Msr1 suppresses the proliferation of LSCs. Using CML mouse model, we show that Msr1 is downregulated by BCR-ABL and this down-regulation is partially restored by Alox5 deletion, and that Msr1 deletion causes acceleration of CML development. Moreover, Msr1 deletion markedly increases LSC function through its effects on cell cycle progression and apoptosis. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-Catenin. Together, these results demonstrate that Msr1 suppresses LSCs and CML development. The enhancement of the tumor suppressor function of Msr1 may be of significance in the development of novel therapeutic strategies for CML.

Arachidonate 15-lipoxygenase is required for chronic myeloid leukemia stem cell survival

Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.

Functional Ramifications for the Loss of P-Selectin Expression on Hematopoietic and Leukemic Stem Cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome -positive (Ph+) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.

Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC)-driven neoplasia characterized by expression of the constitutively active tyrosine kinase BCR/Abl. CML therapy based on tyrosine kinase inhibitors (TKIs) is highly effective in inducing remission but not in targeting leukemia stem cells (LSCs), which sustain minimal residual disease and are responsible for CML relapse following discontinuation of treatment. The identification of molecules capable of targeting LSCs appears therefore of primary importance to aim at CML eradication. LSCs home in bone marrow areas at low oxygen tension, where HSCs are physiologically hosted. This study addresses the effects of pharmacological inhibition of hypoxia-inducible factor-1 (HIF-1), a critical regulator of LSC survival, on the maintenance of CML stem cell potential. We found that the HIF-1 inhibitor acriflavine (ACF) decreased survival and growth of CML cells. These effects were paralleled by decreased expression of c-Myc and stemness-related genes. Using different in vitro stem cell assays, we showed that ACF, but not TKIs, targets the stem cell potential of CML cells, including primary cells explanted from 12 CML patients. Moreover, in a murine CML model, ACF decreased leukemia development and reduced LSC maintenance. Importantly, ACF exhibited significantly less-severe effects on non-CML hematopoietic cells in vitro and in vivo. Thus, we propose ACF, a US Food and Drug Administration (FDA)-approved drug for nononcological use in humans, as a novel therapeutic approach to prevent CML relapse and, in combination with TKIs, enhance induction of remission.

LSK Derived LSK– Cells Have a High Apoptotic Rate Related to Survival Regulation of Hematopoietic and Leukemic Stem Cells

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin−Sca-1+c-Kit- (LSK−) cell population derived from HSC-containing Lin−Sca-1+c-Kit+ (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK− population. In contrast, the LSK− population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK− population. The transition of LSK to LSK− cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK− cells in the regulation of LSK cells and LSCs.

Targeting PFKFB3 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitor

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) remains a challenge for curing the disease in chronic myeloid leukemia (CML) patients as leukemia cells may survive through BCR-ABL kinase activity-independent signal pathways. To gain insight into BCR-ABL kinase activity-independent mechanisms, we performed an initial bioinformatics screen and followed by a quantitative PCR screen of genes that were elevated in CML samples. A total of 33 candidate genes were identified to be highly expressed in TKIs resistant patients. Among those genes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), controlling the limiting step of glycolysis, was found to be strongly associated with TKIs resistance. PFKFB3 knockdown or pharmacological inhibition of its kinase activity markedly enhanced the sensitivity of CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 inhibited CML cells growth and significantly prolonged the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment showed that the Ets transcription factor PU.1 regulated the elevated expression of PFKFB3 in TKIs-resistant CML cells. Therefore, our results showed that targeting PFKFB3 sensitizes CML cells to TKIs and PFKFB3 may be a potential BCR-ABL kinase activity-independent mechanism in CML.

Targeting the Extracellular Signal-Regulated Kinase 5 Pathway to Suppress Human Chronic Myeloid Leukemia Stem Cells

Summary Tyrosine kinase inhibitors (TKi) are effective against chronic myeloid leukemia (CML), but their inefficacy on leukemia stem cells (LSCs) may lead to relapse. To identify new druggable targets alternative to BCR/ABL, we investigated the role of the MEK5/ERK5 pathway in LSC maintenance in low oxygen, a feature of bone marrow stem cell niches. We found that MEK5/ERK5 pathway inhibition reduced the growth of CML patient-derived cells and cell lines in vitro and the number of leukemic cells in vivo. Treatment in vitro of primary CML cells with MEK5/ERK5 inhibitors, but not TKi, strikingly reduced culture repopulation ability (CRA), serial colony formation ability, long-term culture-initiating cells (LTC-ICs), and CD26-expressing cells. Importantly, MEK5/ERK5 inhibition was effective on CML cells regardless of the presence or absence of imatinib, and did not reduce CRA or LTC-ICs of normal CD34+ cells. Thus, targeting MEK/ERK5 may represent an innovative therapeutic approach to suppress CML progenitor/stem cells.