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Purpose:Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis.Experimental Design:A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor).Results:JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo.Conclusions:Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

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PDF file, 421K, Supplementary Figure 1: Specificity of imatinib, dasatinib, PPY-A, BAW667, and a SCF-blocking antibody on Mo7ep210BCR-ABL1 cells. Supplementary Figure 2: Comparison of CFU-GM colony formation upon sole BCR-ABL1 inhibition (PPY-A), sole KIT inhibition or dual BCR-ABL1/KIT inhibition. Supplementary Figure 3: Inhibition of KIT in human cells through use of a lentiviral vector for simultaneous expression of shKIT and GFP . Supplementary Figure 4: Analysis of CML CFU-GM colony growth following removal of the individual cytokines. Supplementary Figure 5: BCR-ABL1 and KIT signaling influence activation of AKT associated with a reduction of Foxo3A. Supplementary Table 1. Activity profile of BAW667.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.

26S proteasome non-ATPase subunits 1 (PSMD1) and 3 (PSMD3) were recently identified as prognostic biomarkers and potential therapeutic targets in chronic myeloid leukemia (CML) and multiple solid tumors. In the present study, we analyzed the expression of 19S proteasome subunits in acute myeloid leukemia (AML) patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) gene and assessed their impact on overall survival (OS). High levels of PSMD3 but not PSMD1 expression correlated with a worse OS in FLT3-mutated AML. Consistent with an oncogenic role for PSMD3 in AML, shRNA-mediated PSMD3 knockdown impaired colony formation of FLT3+ AML cell lines, which correlated with increased OS in xenograft models. While PSMD3 regulated nuclear factor-kappa B (NF-κB) transcriptional activity in CML, we did not observe similar effects in FLT3+ AML cells. Rather, proteomics analyses suggested a role for PSMD3 in neutrophil degranulation and energy metabolism. Finally, we identified additional PSMD subunits that are upregulated in AML patients with mutated versus wild-type FLT3, which correlated with worse outcomes. These findings suggest that different components of the 19S regulatory complex of the 26S proteasome can have indications for OS and may serve as prognostic biomarkers in AML and other types of cancers.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have revolutionized therapy for chronic myeloid leukemia (CML), paving the way for clinical development in other diseases. Despite success, targeting leukemic stem cells and overcoming drug resistance remain challenges for curative cancer therapy. To identify drivers of kinase-independent TKI resistance in CML, we performed genome-wide expression analyses on TKI-resistant versus sensitive CML cell lines, revealing a nuclear factor-kappa B (NF-κB) expression signature. Nucleocytoplasmic fractionation and luciferase reporter assays confirmed increased NF-κB activity in the nucleus of TKI-resistant versus sensitive CML cell lines and CD34+ patient samples. Two genes that were upregulated in TKI-resistant CML cells were proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), both members of the 19S regulatory complex in the 26S proteasome. PSMD1 and PSMD3 were also identified as survival-critical genes in a published small hairpin RNA library screen of TKI resistance. We observed markedly higher levels of PSMD1 and PSMD3 mRNA in CML patients who had progressed to the blast phase compared with the chronic phase of the disease. Knockdown of PSMD1 or PSMD3 protein correlated with reduced survival and increased apoptosis in CML cells, but not in normal cord blood CD34+ progenitors. Luciferase reporter assays and immunoblot analyses demonstrated that PSMD1 and PSMD3 promote NF-κB protein expression in CML, and that signal transducer and activator of transcription 3 (STAT3) further activates NF-κB in scenarios of TKI resistance. Our data identify NF-κB as a transcriptional driver in TKI resistance, and implicate PSMD1 and PSMD3 as plausible therapeutic targets worthy of future investigation in CML and possibly other malignancies.

Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3+, CD14+, and CD34+ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 107 (range, 1.62 × 105–2.52 × 108), and the median yield of CD34+ cells was 1.40 × 106 (range, 3.60 × 105–9.90 × 106). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.

Purpose: Breast cancer (BC) is the leading cause of cancer related deaths in Hispanic women. Hispanics make up an estimated 82% of the U.S./Mexico border, a region of socioeconomic inequity and barriers to healthcare access. Identifying disparities affecting BC incidence and overall survival (OS) is a priority for optimizing care in this medically underserved population. We hypothesized that differences in ethnicity, border proximity, birthplace, BC subtype, and treatments are associated with poor outcomes. Methods: Data from the Texas Cancer Registry was used to construct Kaplan-Meier curves of OS by ethnicity, birthplace, subtype (Luminal A, Luminal B, HER2, Triple Negative (TNBC)), age group (18-39, 40-69, >70 years), and treatments. Other covariates included rurality, insurance status, socioeconomic status, and comorbidities. Crude and adjusted variable effects on OS were analyzed with Mantel Haenszel and Cox regression methods. Results: Hispanic BC patients were diagnosed at a younger age than non-Hispanic whites (NHW) (57.2 versus 61.1 years). Hispanic ethnicity reduced the risk for luminal A and TNBC (HR 0.90, and 0.87, p Citation Format: Alfonso E. Bencomo-Alvarez, Vutha Nhim, Luis Alvarado, Mayra A. Gonzalez, Michelle Kilcoyne, Alok K. Dwivedi, Shrikanth S. Gadad, Anna M. Eiring. Effects of race, ethnicity, and country of origin on incidence and survival in breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C066.

Simple Summary AML is a heterogenous malignancy with a variety of underlying genomic abnormalities. Some of the genetic aberrations in AML have led to the development of specific inhibitors which were approved by the Food and Drug Administration (FDA) and are currently used to treat eligible patients. In this review, we describe five gene mutations for which approved inhibitors have been developed, the response of AML patients to these inhibitors, and the known mechanism(s) of resistance. This review also highlights the significance of developing function-based screens for target discovery in the era of personalized medicine. Abstract Acute myeloid leukemia (AML) is a highly heterogeneous malignancy characterized by the clonal expansion of myeloid stem and progenitor cells in the bone marrow, peripheral blood, and other tissues. AML results from the acquisition of gene mutations or chromosomal abnormalities that induce proliferation or block differentiation of hematopoietic progenitors. A combination of cytogenetic profiling and gene mutation analyses are essential for the proper diagnosis, classification, prognosis, and treatment of AML. In the present review, we provide a summary of genomic abnormalities in AML that have emerged as both markers of disease and therapeutic targets. We discuss the abnormalities of RARA, FLT3, BCL2, IDH1, and IDH2, their significance as therapeutic targets in AML, and how various mechanisms cause resistance to the currently FDA-approved inhibitors. We also discuss the limitations of current genomic approaches for producing a comprehensive picture of the activated signaling pathways at diagnosis or at relapse in AML patients, and how innovative technologies combining genomic and functional methods will improve the discovery of novel therapeutic targets in AML. The ultimate goal is to optimize a personalized medicine approach for AML patients and possibly those with other types of cancers.

Acute myeloid leukemia (AML) is a heterogeneous disease with a poor prognosis and remarkable resistance to chemotherapeutic agents. Understanding resistance mechanisms against currently available drugs helps to recognize the therapeutic obstacles. Various mechanisms of resistance to chemotherapy or targeted inhibitors have been described for AML cells, including a role for the bone marrow niche in both the initiation and persistence of the disease, and in drug resistance of the leukemic stem cell (LSC) population. The BM niche supports LSC survival through direct and indirect interactions among the stromal cells, hematopoietic stem/progenitor cells, and leukemic cells. Additionally, the BM niche mediates changes in metabolic and signal pathway activation due to the acquisition of new mutations or selection and expansion of a minor clone. This review briefly discusses the role of the BM microenvironment and metabolic pathways in resistance to therapy, as discovered through AML clinical studies or cell line and animal models.

Despite significant advances in the treatment of myeloid malignancies, many patients become resistant to therapy and ultimately succumb to their disease. Accumulating evidence over the past several years has suggested that the inadequacy of many leukaemia therapies results from their failure to target the leukaemic stem cell (LSC). For this reason, the LSC population currently represents the most critical target in the treatment of myeloid malignancies. However, while LSCs are ideal targets in the treatment of these diseases, they are also the most difficult population to target. This is due to both their heterogeneity within the LSC population, and also their phenotypic similarities with normal haematopoietic stem cells. This review will highlight the current landscape surrounding LSC biology in myeloid malignancies, with a focus on altered energy metabolism, and how that knowledge is being translated into clinical advances for the treatment of chronic and acute myeloid leukaemia and myelodysplastic syndromes.

Purpose: Myelofibrosis is a hematopoietic stem cell neoplasm characterized by bone marrow reticulin fibrosis, extramedullary hematopoiesis, and frequent transformation to acute myeloid leukemia. Constitutive activation of JAK/STAT signaling through mutations in JAK2, CALR, or MPL is central to myelofibrosis pathogenesis. JAK inhibitors such as ruxolitinib reduce symptoms and improve quality of life, but are not curative and do not prevent leukemic transformation, defining a need to identify better therapeutic targets in myelofibrosis. Experimental Design: A short hairpin RNA library screening was performed on JAK2V617F-mutant HEL cells. Nuclear–cytoplasmic transport (NCT) genes including RAN and RANBP2 were among top candidates. JAK2V617F-mutant cell lines, human primary myelofibrosis CD34+ cells, and a retroviral JAK2V617F-driven myeloproliferative neoplasms mouse model were used to determine the effects of inhibiting NCT with selective inhibitors of nuclear export compounds KPT-330 (selinexor) or KPT-8602 (eltanexor). Results: JAK2V617F-mutant HEL, SET-2, and HEL cells resistant to JAK inhibition are exquisitely sensitive to RAN knockdown or pharmacologic inhibition by KPT-330 or KPT-8602. Inhibition of NCT selectively decreased viable cells and colony formation by myelofibrosis compared with cord blood CD34+ cells and enhanced ruxolitinib-mediated growth inhibition and apoptosis, both in newly diagnosed and ruxolitinib-exposed myelofibrosis cells. Inhibition of NCT in myelofibrosis CD34+ cells led to nuclear accumulation of p53. KPT-330 in combination with ruxolitinib-normalized white blood cells, hematocrit, spleen size, and architecture, and selectively reduced JAK2V617F-mutant cells in vivo. Conclusions: Our data implicate NCT as a potential therapeutic target in myelofibrosis and provide a rationale for clinical evaluation in ruxolitinib-exposed patients with myelofibrosis.

Background: Population-based studies on Hodgkin’s lymphoma (HL) have shown reduced survival in Hispanics and non-Hispanic Blacks compared with non-Hispanic Whites. To better understand the factors contributing to this outcome discrepancy, we retrospectively reviewed the charts of patients with HL diagnosed and treated at a single institution located along the Texas–Mexico border. Patients and Methods: We performed a retrospective chart review of all patients with HL treated at our institution over an 8-year period (2011-2018). The International Prognostic Score was calculated for all patients and results of positron-emission tomography (PET) scans (interim and end of treatment) were also recorded. Variables analyzed included tumor-related findings (stage, subtype of HL), treatment history (chemotherapy regimen including number of cycles, dose intensity and radiation treatments) and neutrophil to lymphocyte ratio. Quantitative variables were described using median, interquartile range, minimum and maximum observations. Categorical variables were described using frequency and proportions. Kaplan–Meier curves were used to show relapse-free survival. Results: A total of 24 patients were treated in the time frame, of whom 23 were Hispanic. All were treated with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) or an ABVD-like regimen. Dose intensity for chemotherapy exceeded 90%. After a median follow-up of 43 months, the relapse rate was 45.8%. Positive and negative predictive values for interim PET (0% and 50%) and end of therapy PET (80% and 58%) were suboptimal to allow for a PET-adapted therapeutic approach. Conclusion: Hispanics have a high relapse rate following ABVD which is not fully explained by universally accepted prognostic factors. Performance of PET scan in predicting outcomes of HL needs to be further studied and optimized before adopting a PET-adapted treatment paradigm for underserved Hispanic populations.

BACKGROUND The Texas/Chihuahua (US/Mexico) border is a medically underserved region with many reported barriers for health care access. Although Hispanic ethnicity is associated with health disparities for many different diseases, the population-based estimates of incidence and survival for patients with blood cancer along the border are unknown. The authors hypothesized that Hispanic ethnicity and border proximity is associated with poor blood cancer outcomes. METHODS Data from the Texas Cancer Registry (1995-2016) were used to investigate the primary exposures of patient ethnicity (Hispanic vs non-Hispanic) and geographic location (border vs non-border). Other confounders and covariates included sex, age, year of diagnosis, rurality, insurance status, poverty indicators, and comorbidities. The Mantel-Haenszel method and Cox regression analyses were used to determine adjusted effects of ethnicity and border proximity on the relative risk (RR) and survival of patients with different blood cancer types. RESULTS Hispanic patients were diagnosed at a younger age than non-Hispanic patients and presented with increased comorbidities. Whereas non-Hispanics had a higher incidence of developing blood cancer compared with Hispanics overall, Hispanics demonstrated a higher incidence of acute lymphoblastic leukemia (RR, 1.92; 95% CI, 1.79-2.08; P < .001) with worse outcomes. Hispanics from the Texas/Chihuahua border demonstrated a higher incidence of chronic myeloid leukemia (RR, 1.28; 95% CI, 1.07-1.51; P = .02) and acute myeloid leukemia (RR, 1.17; 95% CI, 1.04-1.33; P = .0009) compared with Hispanics living elsewhere in Texas. CONCLUSIONS Hispanic ethnicity and border proximity were associated with a poor presentation and an adverse prognosis despite the younger age of diagnosis. Future studies should explore differences in disease biology and treatment strategies that could drive these regional disparities.

FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.

Background: Breast cancer (BC) is the leading cause of cancer related deaths in Hispanic women. Hispanics make up an estimated 82% of the US/Mexico border, a region characterized by socioeconomic inequity and barriers to healthcare access. Identifying disparities associated with BC incidence and overall survival (OS) is a priority for allocating resources and optimizing care in this medically underserved population. We hypothesized that differences in Hispanics and Non-Hispanic Whites (NHW), proximity to the border, BC subtype, and treatment are associated with poor outcomes. Methods: BC data was obtained from the Texas Cancer Registry (1996-2016). Kaplan-Meier curves of OS by ethnicity, location (border, non-border), subtype (ER+, PR+, HER2+, Triple Negative (TN)), age group (18-39, 40-69, >70 years), and treatments were constructed. Other covariates included rurality, insurance status, poverty indicators, and comorbidities. Adjustment of these variables for effect on relative risk and OS were assessed with Mantel-Haenszel and Cox regression methods. Results: Univariate Cox analysis noted significantly higher Hazard Ratios (HR) for age >65 years, HER2+ and TN subtype (HR 1.76 and 2.21, p70 years, p Citation Format: Vutha Nhim, Alfonso E. Bencomo-Alvarez, Alok K. Dwivedi, Shrikanth S. Gadad, Anna M. Eiring. Border differences on breast cancer incidence and survival between non-Hispanic white and Hispanic patients: A Texas population-based study [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-200.

Ever since the ubiquitin proteasome system was characterized, efforts have been made to manipulate its function to abrogate the progression of cancer. As a result, the anticancer drugs bortezomib, carfilzomib, and ixazomib targeting the 26S proteasome were developed to treat multiple myeloma, mantle cell lymphoma, diffuse large B-cell lymphoma, colorectal cancer, and thyroid carcinoma. Despite the success demonstrated by these treatments, adverse side effects and drug resistance are prominent, raising the need for alternative therapeutic options. The 26S proteasome is composed of two 19S regulatory subunits and a 20S catalytic core, leaving ample opportunity for new targets. We recently demonstrated that knockdown of the 19S regulatory subunits, proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), resulted in increased apoptosis of tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cells, but had no effect on normal cord blood controls, suggesting they may be good targets for therapy. Therefore, we hypothesized that PSMD1 and PSMD3 are potential targets for anticancer therapeutics and that their relevance stretches beyond CML to other types of cancers. In the present study, we analyzed PSMD1 and PSMD3 expression in other types of cancers versus normal controls using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), comparing expression with overall survival (OS). Data were analyzed using UALCAN (http://ualcan.path.uab.edu) and GEPIA2 (http://gepia2.cancer-pku.cn/). At the mRNA level, PSMD1 was found to be overexpressed in 14/24 (58%) TCGA cancers compared with normal controls. This was confirmed at the protein level for breast (p=4.2e-4), colon (p=7.1e-20), clear cell renal carcinoma (p=1.8e-4), and endometrial cancer (p=2.0e-22). PSMD3 was found upregulated at the mRNA level in 18/24 (75%) TCGA cancers, which was confirmed at the protein level in breast (p=8.3e-4), colon (p=1.0e-27), ovarian (p=4.2e-6), clear cell renal carcinoma (p=4.0e-19), and endometrial cancer (p=1.5e-19). Upon correlation of PSMD1 expression with OS, we saw that when overexpressed, individuals did significantly worse with adrenocortical carcinoma, lower grade glioma, lung adenocarcinoma, mesothelioma, and uveal melanoma. In contrast, diffuse large B cell lymphoma (DLBCL) and stomach adenocarcinoma patients with lower PSMD1 expression had a worse OS. Similarly, patients with high levels of PSMD3 mRNA expression had a significantly worse OS in kidney chromophobe, skin cutaneous melanoma, uveal melanoma, and mesothelioma. Our analysis revealed that differential expression of PSMD1 and PSMD3 is correlated with survival in several different cancer types. Future directions will identify PSMD1 and PSMD3 post-translational modifications that may be novel targets for anticancer therapeutics. In conclusion, we highlight PSMD1 and PSMD3 as potential therapeutic targets for the development of novel proteasome inhibitors to treat cancer patients with less toxicity. Citation Format: Andres J. Rubio, Alfonso E. Bencomo-Alvarez, James E. Young, Vanessa V. Velazquez, Anna M. Eiring. PSMD1 and PSMD3 as putative targets for cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P150.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. Disclosures No relevant conflicts of interest to declare.

Background Chronic phase chronic myeloid leukemia (CP-CML) is characterized by overproduction of differentiating myeloid cells, while blast phase CML (BP-CML) cells exhibit a differentiation block. Tyrosine kinase inhibitors (TKIs) are effective in CP-CML, but resistance is common in BP-CML and can occur without explanatory BCR-ABL1 kinase mutations (BCR-ABL1-independent resistance). Similarly, CML stem/progenitor cells (LSPCs) are insensitive to TKIs, and residual leukemia persists in the majority of CML patients on TKI therapy. We previously reported overlap between the transcriptomes of CD34 + cells from BP-CML and TKI-naïve CP-CML patients with primary TKI resistance, pointing to commonalities between LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML. Results To identify common mechanisms, we performed a meta-analysis of published CML transcriptomes. We identified a small set of genes with consistently low expression in LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML, including Membrane Spanning 4-Domains A3 (MS4A3), a signaling protein previously reported to inhibit hematopoietic cell cycle progression. Low MS4A3 in CD34 + cells from TKI-naïve CP-CML patients was associated with shorter survival on subsequent TKI therapy, suggesting that MS4A3 governs TKI response. To understand the function of MS4A3, we lentivirally introduced MS4A3 shRNA or an MS4A3 expression vector into CML CD34 + LSPCs. MS4A3 knockdown increased clonogenicity and imatinib resistance, while ectopic MS4A3 expression had opposite effects. MS4A3 KD also increased LSPC persistence ex vivo in LTC-IC assays, and in vivo in NSG mice xenografts, while modulating MS4A3 expression had no effect on normal CD34 + cells. We next generated Ms4a3+/+│-/-; Scl-tTA+; TRE-BCR-ABL1+ compound transgenic mice. Upon BCR-ABL1 induction, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice developed leukocytosis comparable to Ms4a3+/+ controls. However, BM of Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice showed increased short-term HSCs and multipotent progenitor cells, and reduced granulocyte-macrophage progenitors. When Lin - BM cells from leukemic mice were transplanted into irradiated recipients, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ cells showed increased engraftment and myeloid leukocytosis, validating our observations in human cells. To determine how MS4A3 is downregulated in CML, we expressed BCR-ABL1in 32D-cl3 cells. p210 BCR-ABL1 drastically reduced Ms4a3 expression, while kinase-inactive p210 BCR-ABL1-K271R had no effect. Moreover, we found that suppression of C/EBPε by MECOM reduces MS4A3, consistent with previous reports of MECOM as a driver of TKI resistance and progression to BP. Treatment of CML CD34 + cells with a library of epigenetic pathway inhibitors revealed that MS4A3 is suppressed by both DNA methylation and PRC2/EZH2-mediated H3K27 trimethylation, which was confirmed by patch-PCR and ChIPseq. These data indicate that multi-levelled mechanisms cooperate in the suppression of MS4A3 in CML. To determine how MS4A3 regulates clonogenicity and TKI response, we expressed MS4A3-EGFP fusion protein in LAMA-84 CML cells. We found that MS4A3 resides on the plasma membrane and in endosomes. Surface protein biotin labelling and tandem mass spectrometry ± MS4A3 KD showed that MS4A3 controls endocytosis of membrane proteins, including common β chain (βc) cytokine receptors. Specifically, MS4A3 promotes endocytosis of βc cytokine receptors upon GM-CSF/IL-3 stimulation of primary LSPCs and enhances downstream signaling and differentiation, suggesting that restoring MS4A3 expression has therapeutic efficacy. To test this, we manufactured a prototype MS4A3 protein-loaded liposomal nanoparticle (NP) using coating with the CD34 CD62L for targeted delivery to CD34 + cells. Compared to MS4A3-free NPs, MS4A3 NPs increased CD34 +CD38 + and CD34 -CD38 + at the expense of CD34 +CD38 - cells, reduced clonogenicity, and increased sensitivity to TKIs, mimicking ectopic MS4A3 expression. Conclusion MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of primitive LSPCs and BP-CML cells. We posit that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation to maintain a primitive, TKI-insensitive state. MS4A3 re-expression or delivery of ectopic MS4A3 may eliminate LSPCs. Figure 1 Figure 1. Disclosures Druker: Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; EnLiven: Consultancy, Research Funding; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aileron: Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Nemucore Medical Innovations, Inc.: Consultancy; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Research Funding; Recludix Pharma, Inc.: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Agios: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Schrodinger: Research Funding. Oehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy. Radich: BMS: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Deininger: Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Ever since the ubiquitin proteasome system was characterized, efforts have been made to manipulate its function to abrogate the progression of cancer. As a result, the anti-cancer drugs bortezomib, carfilzomib, and ixazomib targeting the 26S proteasome were developed to treat multiple myeloma, mantle cell lymphoma, and diffuse large B-cell lymphoma, among others. Despite success, adverse side effects and drug resistance are prominent, raising the need for alternative therapeutic options. We recently demonstrated that knockdown of the 19S regulatory components, 26S proteasome non-ATPase subunits 1 (PSMD1) and 3 (PSMD3), resulted in increased apoptosis of chronic myeloid leukemia (CML) cells, but had no effect on normal controls, suggesting they may be good targets for therapy. Therefore, we hypothesized that PSMD1 and PSMD3 are potential targets for anti-cancer therapeutics and that their relevance stretches beyond CML to other types of cancers. In the present study, we analyzed PSMD1 and PSMD3 mRNA and protein expression in cancerous tissue versus normal controls using data from The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), comparing expression with overall survival. Altogether, our data suggest that PSMD1 and PSMD3 may be novel putative targets for cancer prognosis and therapy that are worthy of future investigation.

FLT3-ITD+ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.

Cancer is a challenging, multifaceted disease that involves a combination of biological and nonbiological factors. Aside from COVID-19, cancer is the second leading cause of death in the United States and the first among Hispanic Americans. The Hispanic population is the largest minority group in the United States, which is rapidly growing in size. Unfortunately, U.S. Hispanics and other minority groups experience many different health disparities, resulting in poor survival outcomes and a reduced quality of life. Factors such as genomic mutations, lower socioeconomic status, lack of education, reduced access to health care, comorbidities, and environmental factors all contribute to these health-care inequalities. In the context of blood cancer health disparities, Hispanic patients are often diagnosed at a younger age and have worse outcomes compared with non-Hispanic individuals. In this commentary, we highlight the existing knowledge about cancer health disparities in the Hispanic population, with a focus on chronic and acute leukemia. In our experience at the U.S./Mexican border, analysis of several different blood cancers demonstrated that younger Hispanic patients with acute lymphoid or myeloid leukemia have higher incidence rates and worse prognoses. A combined approach, involving improved health-care access and better knowledge of the underlying factors, will allow for more timely diagnoses and the development of intervention strategies aimed at reducing or eliminating the disparities.

Rapid conversion of chronic myeloid leukemia to chronic myelomonocytic leukemia in a patient on imatinib therapy

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾ 10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾ 2 ARCH genes and 52% had ⩾ 7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. However, resistance is a clinical problem, and TKIs do not target CML leukemic stem cells (LSC), which are independent from BCR-ABL1 kinase activity. To understand mechanisms driving BCR-ABL1-independent resistance, we analyzed the transcriptional signature of TKI-naïve CD34+ cells from patients with or without a response to imatinib after 12 months of therapy (McWeeney et al. Blood 2010). Among the genes most profoundly downregulated in patients destined to fail imatinib was G0/G1 switch gene 2 (G0S2) (>3-fold, n=59, p50%) correlated with a longer overall survival (n=30 responders, n=16 non-responders, p=0.036). We next analyzed G0S2 mRNA expression in CD34+ cells from normal cord blood and from primary CD34+ cells lacking BCR-ABL1 kinase domain mutations. G0S2 was 4-fold downregulated in newly diagnosed CML (n=6) compared to normal cord blood (n=5, p3-fold in TKI-resistant (n=2) and BP-CML samples (n=5, p Citation Format: Mayra A. Gonzalez, Alfonso E. Bencomo, Christian Barreto-Vargas, Andres J. Rubio, Idaly M. Olivas, Joshua J. Lara, Anna Senina, Jonathan Ahmann, Katherine T. Varley, Luis F. Jave-Suarez, O'Hare Thomas, Michael W. Deininger, Anna M. Eiring. Role of G0S2 in chronic myeloid leukemia and TKI resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 648.

Introduction: Hispanics represent the largest minority group in the United States (U.S.), with 57.5 million individuals. The majority of Hispanics in the U.S. reside in the Southwest region, and >11 million live in the state of Texas. Health disparities for Hispanic cancer patients have previously been linked to disproportionate poverty and other barriers to optimal healthcare. In the case of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), Hispanics were found to be diagnosed at a younger age and have worse overall survival (OS) compared with non-Hispanic whites (NHWs) (ACS Cancer Facts & Figures for Hispanics/Latinos 2018-2020). However, little is known about incidence and survival for Hispanic blood cancer patients residing at the U.S./Mexico border. Methods: We retrospectively reviewed data from the Texas Cancer Registry for hematologic malignancies diagnosed in the state of Texas between 1995 and 2016, focusing our analysis on chronic and acute leukemia's (both myeloid and lymphoid), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPNs). Survival for Hispanic and NHW groups was compared using the log-rank test, and Cox regression analyses adjusting for age and diagnosis. Differences in age at diagnosis were evaluated using t-tests and generalized linear models. Research was conducted according to a local IRB-approved protocol in accordance with the Declaration of Helsinki. Results: Of the 69,941 cases of hematologic malignancies with available information throughout the state of Texas, 18.29% identified as Hispanic. We found that Hispanic patients were diagnosed at a significantly younger age in all diseases analyzed (p Conclusions: Hispanics are diagnosed at a significantly younger age than NHWs in all blood malignancies analyzed. Hispanic patients with AML, MDS, and CML had significantly better OS compared to NHWs in unadjusted analyses, which could be explained by the reduced age of diagnosis. Hispanics with ALL, AML, or CML diagnosed near the U.S./Mexico border demonstrate worse OS compared with Hispanics diagnosed in other areas of Texas. In age-adjusted analyses, Hispanic patients with ALL or APL have a worse OS compared with NHWs. There appears to be evidence that disparities in outcome by ethnicity may be different in El Paso compared with the rest of Texas. Future Directions: Our data demonstrates blood cancer disparities present in our region. Further study is required to identify factors responsible for the disparity in OS, and to identify ways to address it. Citation Format: Alfonso E. Bencomo, Andres J. Rubio, Mayra A. Gonzalez, Idaly M. Olivas, Joshua J. Lara, Kiran Ghimire, Osvaldo Padilla, Angelica Padilla, Nawar Hakim, Attilio Orazi, Javier Corral, Alexander Philipovskiy, Sumit Gaur, Anna M. Eiring. Retrospective study of incidence and survival for patients with hematological malignancies residing at the U.S./Mexico border [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4343.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are effective at eliminating most BCR-ABL1+ cells in chronic myeloid leukemia (CML), but do not target CML leukemic stem cells (LSCs), which are independent of BCR-ABL1 kinase activity. Our previous work showed that BCR-ABL1-independent resistance is largely driven by STAT3 (Eiring et al. Leukemia 2015). To understand how STAT3 contributes to TKI resistance, we performed RNA sequencing on TKI-sensitive K562-S cells versus TKI-resistant K562-R cells, which demonstrate kinase-independent resistance. Surprisingly, gene set enrichment analysis did not reveal STAT3-mediated transcription (p=1.0), but was reminiscent of TNFα signaling via NF-κB (p=0.024). Nucleocytoplasmic fractionation revealed higher levels of phospho-NF-κB in the nucleus of K562-R vs. K562-S controls, and in CD34+ progenitors from TKI-resistant CML patients (n=3) compared to TKI responders (n=2) and normal individuals (n=2). These data suggest NF-κB may be driving the transcriptional signature of TKI resistance, and implicate non-canonical functions for STAT3. To confirm increased NF-κB transcriptional activity in TKI resistance, K562-S and K562-R cells were transduced with an NF-κB luciferase reporter construct or a scrambled control. K562-R cells demonstrated increased NF-κB reporter activity compared to K562-S cells. To assess whether STAT3 activates NF-κB, reporter cells were co-infected with an inducible lentiviral shRNA targeting STAT3 (shSTAT3). STAT3 knockdown decreased NF-κB reporter activity in TKI-resistant cells, but increased reporter activity in TKI-sensitive controls. These data suggest that STAT3 may cooperate with NF-κB in the setting of TKI resistance. NF-κB and STAT3 have been shown to cooperatively bind to gene promoters of cytokines, specifically IL-6. ELISA assays demonstrated that K562-R cells produce autonomous IL-6, but not TNFα. Since IL-6 is a potent activator of STAT3, we treated TKI-resistant cells with the IL-6 receptor inhibitor, tocilizumab (100 and 1000 ng/ml). As expected, tocilizumab treatment reduced STAT3 Y705 phosphorylation as assessed by immunoblot analysis, but surprisingly had no effect on survival or NF-κB reporter activity in K562-R cells. These data suggest that STAT3 phosphorylation is not required for survival or NF-κB activation in TKI resistance. The role of unphosphorylated STAT3 in TKI resistance is currently being explored. We correlated our RNA sequencing data in TKI-resistant cell lines with microarray data in TKI-resistant patient samples (McWeeney et al. Blood 2010), identifying upregulation of the proteasome components, PSMD1 and PSMD3. qRT-PCR confirmed upregulation of PSMD3 but not PSMD1 in TKI resistance, and only the PSMD3 promoter was bound by NF-κB as assessed by ChIP. Lentiviral shRNAs targeting PSMD3 (shPSMD3) reduced colony formation of K562-S and K562-R cells, which correlated with induction of apoptosis, an increase of global protein ubiquitination, and decreased NF-κB activation, with little effects observed in normal cord blood. Altogether, our data implicate a positive feedback loop involving NF-κB and PSMD3 in TKI resistance and suggest that PSMD3 may be a novel target for treatment of TKI-resistant CML. Citation Format: Andres J. Rubio, Idaly M. Olivas, Alfonso E. Bencomo, Mayra A. Gonzalez, Joshua J. Lara, Rebecca Ellwood, Carme Ripoll-Fiol, Georgios Nteliopoulos, Alistair Reid, Dragana Milojkovic, Jane Apperley, Jamshid Sorouri-Khorashad, Anna M. Eiring. NF-kB associates with tyrosine kinase inhibitor resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 647.

Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CC(mut3), which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CC(mut3) exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CC(mut3) inhibits colony formation by primary CML CD34(+) cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.

Introduction: Hispanics represent the largest minority group in the United States (U.S.), with 57.5 million individuals (18% of the population). Most U.S. Hispanics are of Mexican origin (63.2%), followed by Puerto Rican (9.5%), Cuban (3.9%), Salvadoran (3.8%), and Dominican (3.3%), but distribution varies by state. The majority of Hispanics in the U.S. reside in the Southwest region, and >11 million live in the state of Texas. Cancer is the leading cause of death in the Hispanic population, accounting for 21% of deaths in people of all ages. Health disparities for Hispanic cancer patients have previously been linked to disproportionate poverty and other barriers to optimal healthcare, and in the case of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), Hispanics were found to be diagnosed younger and to have worse overall survival (OS) than Non-Hispanic whites (NHWs) (ACS. Cancer Facts & Figures for Hispanics/Latinos 2018-2020). However, little is known about incidence and survival for Hispanic blood cancer patients residing at the U.S./Mexico border. To understand the impact of Hispanic ethnicity on outcomes for blood cancer patients diagnosed in this area, we examined OS in adult patients with hematologic malignancies throughout the state of Texas compared to Texas Health Service Region (HSR) 10, encompassing El Paso County. Methods: We retrospectively reviewed data available from the Texas Cancer Registry for hematologic malignancies diagnosed in the state of Texas between 1995 and 2016, focusing our analysis on chronic and acute leukemias (both myeloid and lymphoid), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPNs). Survival for Hispanic and NHW groups was compared using the log-rank test, and Cox regression analyses adjusting for age and diagnosis. Differences in age at diagnosis were evaluated using t-tests and generalized linear models. Similar analyses compared Hispanic patients from HSR 10 versus Hispanic patients from the rest of Texas. Research was conducted according to a local Institutional Review Board-approved protocol in accordance with the Declaration of Helsinki. Results: Of the 69,941 cases of hematologic malignancies with available information throughout the state of Texas, 18.29% self-identified as Hispanic. Surprisingly, in unadjusted analyses, Hispanic patients had significantly better OS than NHWs diagnosed with AML (p Conclusions: While Hispanic patients with AML, MDS, and CML had significantly better OS compared to NHWs in Texas as a whole, this could be explained by a significant reduction in the age of diagnosis for Hispanics. However, when comparing across Texas, El Paso Hispanics with ALL, AML, and CML have a worse prognosis than in the rest of the state. There appears to be evidence that disparities in outcome by ethnicity may be different in El Paso compared with the rest of Texas. Further study is required to identify factors responsible for the disparity in OS. Disclosures No relevant conflicts of interest to declare.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. Despite improved survival, resistance is a clinical problem, and TKIs do not target the quiescent CML leukemic stem cell (LSC), meaning that patients must be treated for life at a high economic burden and sometimes with significant side effects. TKI resistance is frequently characterized by mutations in the BCR-ABL1 kinase domain, but they explain only ~50% of clinical TKI failure. The remaining patients have BCR-ABL1-independent resistance, defined as survival despite BCR-ABL1 inhibition. We have previously reported a gene expression signature predictive of TKI failure in CD34+ cells from chronic phase CML patients (McWeeney et al. 2010). This gene expression signature demonstrated significant overlap with signatures of blast phase CML (Zheng et al. 2006), suggesting that similar biological processes may be driving TKI resistance and disease progression. We expanded our analysis, and also found significant overlap between the expression profiles of TKI resistance and quiescent CML LSCs reported by Graham et al. (p=4x10-14) and Cramer Morales et al. (p=7x10-6). These data suggest that there is a core of genes whose expression is consistently associated with multiple scenarios of BCR-ABL1-independent resistance. Our previous work has shown that BCR-ABL1-independent resistance is largely driven by STAT3, and that targeting STAT3 in combination with TKIs restores sensitivity in TKI-resistant CML stem and progenitor cells (Eiring et al. Leukemia 2015). Unexpectedly, gene set enrichment analysis revealed that our gene expression signature predictive of TKI failure (McWeeney et al. 2010) does not reveal a STAT3 transcriptional signature. Similarly, RNA sequencing data on TKI-resistant K562-R cells, which are resistant to TKIs but lack BCR-ABL1 kinase domain mutations, revealed that, while TKI resistance was not associated with a STAT3 transcriptional signature (p=1.0), it was correlated with a signature reminiscent of TNFα signaling via NFκB (p=0.024). Nucleocytoplasmic fractionation revealed higher levels of total- and phospho-NFκB in the nucleus of K562-R versus K562-S cells, and in CD34+progenitors from TKI-resistant CML patients (n=3) compared to TKI responders (n=2) and normal individuals (n=2). These data indicate that NFκB may be driving the gene expression signature associated with BCR-ABL1-independent resistance, and suggest non-canonical functions for STAT3 that go beyond its traditional role as a transcription factor. Citation Format: Anna M. Eiring, Rebecca Ellwood, Carme Ripoll Fiol, Robert K. Hills, Georgios Nteliopoulos, Alistair Reid, Dragana Milojkovic, Jane Apperley, Jamshid Sorouri-Khorashad. Mechanisms of tyrosine kinase inhibitor resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3018.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.

The success of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) depends on the requirement for BCR-ABL1 kinase activity in CML progenitors. However, CML quiescent HSCs are TKI resistant and represent a BCR-ABL1 kinase–independent disease reservoir. Here we have shown that persistence of leukemic HSCs in BM requires inhibition of the tumor suppressor protein phosphatase 2A (PP2A) and expression — but not activity — of the BCR-ABL1 oncogene. Examination of HSCs from CML patients and healthy individuals revealed that PP2A activity was suppressed in CML compared with normal HSCs. TKI-resistant CML quiescent HSCs showed increased levels of BCR-ABL1, but very low kinase activity. BCR-ABL1 expression, but not kinase function, was required for recruitment of JAK2, activation of a JAK2/β-catenin survival/self-renewal pathway, and inhibition of PP2A. PP2A-activating drugs (PADs) markedly reduced survival and self-renewal of CML quiescent HSCs, but not normal quiescent HSCs, through BCR-ABL1 kinase–independent and PP2A-mediated inhibition of JAK2 and β-catenin. This led to suppression of human leukemic, but not normal, HSC/progenitor survival in BM xenografts and interference with long-term maintenance of BCR-ABL1–positive HSCs in serial transplantation assays. Targeting the JAK2/PP2A/β-catenin network in quiescent HSCs with PADs (e.g., FTY720) has the potential to treat TKI-refractory CML and relieve lifelong patient dependence on TKIs.

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

Pharmacologic blockade of the activation of signal transducer and activator of transcription 3 (STAT3) in tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML) cell lines characterized by kinase-independent resistance was shown to re-sensitize CML cells to TKI therapy, suggesting that STAT3 inhibitors in combination with TKIs are an effective combinatorial therapeutic for the treatment of CML. Benzoic acid- and hydroxamic acid-based STAT3 inhibitors SH-4-054 and SH-5-007, developed previously in our laboratory, demonstrated promising activity against these resistant CML cell lines. However, pharmacokinetic studies in murine models (CD-1 mice) revealed that both SH-4-054 and SH-5-007 are susceptible to glutathione conjugation at the para position of the pentafluorophenyl group via nucleophilic aromatic substitution (SN Ar). To determine whether the electrophilicity of the pentafluorophenyl sulfonamide could be tempered, an in-depth structure-activity relationship (SAR) study of the SH-4-054 scaffold was conducted. These studies revealed that AM-1-124, possessing a 2,3,5,6-tetrafluorophenylsulfonamide group, retained STAT3 protein affinity (Ki =15 μm), as well as selectivity over STAT1 (Ki >250 μm). Moreover, in both hepatocytes and in in vivo pharmacokinetic studies (CD-1 mice), AM-1-124 was found to be dramatically more stable than SH-4-054 (t1/2 =1.42 h cf. 10 min, respectively). AM-1-124 is a promising STAT3-targeting inhibitor with demonstrated bioavailability, suitable for evaluation in preclinical cancer models.